Methods and compositions for manipulating nucleic acids

ABSTRACT

The present disclosure provides methods, compositions and kits as well as systems for manipulating nucleic acids, including implementing isothermal amplification, such as recombinase-polymerase amplification (RPA), of a nucleic acid template using a pre-seeded solid support. Provided are rapid and efficient methods for generating template nucleic acid molecules comprising specific nucleotide sequence bound to solid support. Such methods can be used, for example, in manipulating nucleic acids in preparation for analysis methods that utilize monoclonal populations of nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/582,597, filed Nov. 7, 2017, entitled “METHODS ANDCOMPOSITIONS FOR ISOTHERMAL NUCLEIC ACID AMPLIFICATION” and to U.S.Provisional Application No. 62/719,078, filed Aug. 16, 2018, entitled“SYSTEM AND METHOD FOR PREPARING A SEQUENCING DEVICE,” the disclosure ofeach of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“LT01179_ST25.txt” created on Feb. 27, 2019 and is 2,000 bytes in size.The sequence listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

INCORPORATION BY REFERENCE

The disclosures of any patents, patent applications, and publicationscited herein are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present disclosure relates generally to methods for manipulating andanalyzing nucleic acids, and compositions and kits for doing the same.

BACKGROUND

Manipulation of nucleic acid samples, such as, for example, nucleic acidamplification, is very useful in molecular biology and has wideapplicability in practically every aspect of biology, therapeutics,diagnostics, forensics and research. Increasingly, biological andmedical research is turning to nucleic acid sequencing for enhancingbiological studies and medicine. For example, biologists and zoologistsare turning to sequencing to study the migration of animals, theevolution of species, and the origins of traits. The medical communityis using sequencing for studying the origins of disease, sensitivity tomedicines, and the origins of infection. The use of sequencing can belimited by insufficient quantity and/or quality of the nucleic acids ina sample. Additionally, sequencing has historically been an expensiveprocess, thus limiting its practice.

Generally, to increase the amount of nucleic acid available foranalysis, amplicons are generated from a nucleic acid molecule using oneor more primers, where the amplicons are complementary to all or aportion of the template from which they were generated. Multiplexedamplification can also streamline processes and reduce overheads. Forsome downstream applications, monoclonality is desirable becausedifferent characteristics of diverse nucleic acid molecules within apolyclonal population can complicate the interpretation of assay data.In instances where a monoclonal population of nucleic acids is desiredfor use in analytical methods, challenges also exist in containingmonoclonal nucleic acid populations and keeping them segregated andfree, or relatively free, of significant contamination by other nucleicacids that are not identical to those in the monoclonal population. Thisis particularly an issue when attempting to conduct analysis, e.g.,sequencing, of multiple samples of different nucleic acids in ahigh-throughput, automated, cost-efficient manner. In nucleic acidsequencing applications, the presence of polyclonal populations cancomplicate the interpretation of sequencing data; however, manysequencing systems are not sensitive enough to detect nucleotidesequence data from a single template nucleic acid molecule, thusamplification of template nucleic acid molecules prior to sequencing isnecessary.

One example of such amplification is recombinase-polymeraseamplification (RPA), which is a DNA amplification process that utilizesenzymes to bind oligonucleotide primers to their complementary partnersin duplex DNA followed by isothermal amplification. RPA offers a numberof advantages over traditional methods of amplification including, e.g.,lack of need for initial thermal or chemical melting, ability to operateat low constant temperatures without absolute temperature control, and areaction mixture (e.g., lacking a target polynucleotide) can be storedin a dried condition. These advantages demonstrate that RPA is apowerful and convenient tool for amplifying nucleic acid molecules.However, attempts at using RPA to prepare template nucleic acidmolecules prior to sequencing have resulted in undesirable polyclonalpopulations and/or insufficient preparations. Thus, there remains a needfor improved methods generating improved preparations of templatenucleic acid molecules for molecular characterization, e.g., sequencing.

SUMMARY

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses for performing themethods, of manipulating nucleic acids.

In some embodiments, rapid and efficient methods for generating anucleic acid molecule comprising a specific nucleotide sequence areprovided herein. Such methods can be used, for example, in manipulatingnucleic acids in preparation for analysis in methods that utilizemonoclonal populations of nucleic acids. Some embodiments of clonalamplification methods disclosed herein for generating monoclonal nucleicacid populations begin with a confined nucleic acid molecule template.Methods of confining nucleic acid molecules provided herein includecapture of single nucleic acid molecules through the binding of aspecific nucleotide sequence common to different nucleic acids to beamplified and analyzed. To generate different nucleic acid moleculeshaving a common specific nucleotide that can be used for ease ofconfinement, one method disclosed herein includes (a) obtaining apopulation of nucleic acid molecules, such as, for example, adouble-stranded, adapter-containing DNA library, in which each moleculecontains, with respect to one of the strands of the molecule, a firstsequence of contiguous nucleotides at the 5′ end of the molecule, asecond sequence of contiguous nucleotides at the 3′ end of the moleculeand a third nucleotide sequence positioned between the first and secondnucleotide sequences, wherein the first and second nucleotide sequencesare different, the first nucleotide sequences of the nucleic acidmolecules are substantially identical and the second nucleotidesequences of the nucleic acid molecules are substantially identical; (b)subjecting the population of nucleic acid molecules to a cycle ofnucleic acid amplification in the presence of a forward primercontaining an oligonucleotide sequence substantially identical to thefirst nucleotide sequence and a reverse primer containing anoligonucleotide sequence complementary to a subsequence of the 5′ end ofthe second nucleotide sequence that is linked at the 3′ end of thesubsequence to a fourth nucleotide sequence that is not complementary tothe second nucleotide sequence; and (c) subjecting the products of thecycle of amplification of step (b) to a cycle of amplification in thepresence of the forward and reverse primers to generate multipledifferent nucleic acid products in which only one of the productscomprises a sequence of nucleotides complementary to the fourthnucleotide sequence. In some embodiments of this method, the reverseprimer includes a nucleotide sequence complementary to the 5′ end of thesecond nucleotide sequence but does not contain a nucleotide sequencecomplementary to the 3′ end of the second nucleotide sequence.

Other embodiments of methods provided herein of generating differentnucleic acid molecules having a common specific nucleotide sequence thatcan be used, for example, for ease of confinement include (a) obtaininga population of nucleic acid molecules, such as, for example, adouble-stranded, adapter-containing DNA library, in which each moleculecontains, with respect to one of the strands of the molecule, a firstsequence of contiguous nucleotides at the 5′ end of the molecule, asecond sequence of contiguous nucleotides at the 3′ end of the moleculeand a third nucleotide sequence positioned between the first and secondnucleotide sequences, wherein the first and second nucleotide sequencesare different, the first nucleotide sequences of the nucleic acidmolecules are substantially identical and the second nucleotidesequences of the nucleic acid molecules are substantially identical; and(b) subjecting the population of nucleic acid molecules to two or morecycles of nucleic acid amplification in the presence of one or moreforward primers containing an oligonucleotide sequence substantiallyidentical to the first nucleotide sequence and a reverse primer that isblocked at the 3′ end and contains an oligonucleotide sequencecomplementary to the second nucleotide sequence that is linked at the 5′end of the oligonucleotide sequence to a fourth nucleotide sequence thatis not complementary to the second nucleotide sequence to generatenucleic acid products in which substantially all of the productscomprise a sequence of nucleotides complementary to the fourthnucleotide sequence.

In some embodiments, provided herein are methods for generating one ormore templated supports, such as, for example, solid supports. Suchmethods include, for example: a) forming a templating reaction mixtureby combining one or more pre-seeded supports, nucleotides, arecombinase, and a polymerase, wherein the one or more pre-seeded solidsupports include a population of attached substantially identical firstprimers and have substantially monoclonal template nucleic acidmolecules attached thereto, wherein the one or more pre-seeded solidsupports are formed in a separate pre-seeding reaction that precedes atemplating reaction, wherein the substantially monoclonal templatenucleic acid molecules include a proximal segment including the firstprimer, which, in some embodiments, does not include 100 or moreidentical nucleotides, wherein the proximal segment attaches a templatenucleic acid segment to a pre-seeded solid support, and wherein thepre-seeded solid supports further include attached first primers thatare attached to the pre-seeded solid support and are not bound totemplate nucleic acid molecules, wherein the templating reaction mixturefurther includes a population of substantially identical soluble secondprimers, and wherein the template nucleic acid molecules include aprimer binding site for the second primer at or near the terminal endthat is opposite the proximal segment; and b) performing the templatingreaction by adding a cation to the templating reaction mixture andincubating the reaction mixture under isothermal conditions for at least10 minutes to amplify the template nucleic acid molecules in atemplating reaction to generate one or more templated solid supports,wherein each of the templated solid supports includes at least 100,000substantially monoclonal template nucleic acid molecules, and whereintemplate nucleic acid molecules are not present in solution in thereaction mixture when the templating reaction is initiated, therebygenerating one or more templated solid supports. In some embodiments,the one or more templated solid supports are used in a sequencingreaction to determine the sequences of the template nucleic acidmolecules. In further embodiments, the templated solid supports aretemplated beads and the sequencing include distributing the beads inwells of a solid support before a sequencing reaction is performed. Insome embodiments, the one or more templated solid supports include afirst templated solid support that is attached with a substantiallymonoclonal population of template nucleic acid molecules having a firstsequence, and at least one other templated solid support that isattached with a substantially monoclonal population of template nucleicacid molecules having a second sequence, wherein the sequence of thefirst attached template nucleic acid molecules differ from the sequenceof the second attached template nucleic acid molecules. In furtherembodiments, the substantially monoclonal template nucleic acidmolecules attached to each pre-seeded solid support include at least 70%of all template nucleic acid molecules attached to each pre-seeded solidsupport. In some embodiments, the templating reaction mixture includes apopulation of pre-seeded solid supports. In further embodiments, eachpre-seeded solid support of the population of solid supports has between10 and 50,000 substantially monoclonal template nucleic acid moleculesattached thereto and the one or more solid supports are beads. In someembodiments, the pre-seeding reaction mixture and/or the templatingreaction mixture further includes a recombinase-accessory protein. Infurther embodiments, the recombinase-accessory protein is asingle-stranded binding protein and/or a recombinase-loading protein. Insome embodiments, the templating reaction mixture and/or the pre-seedingreaction mixture is incubated at a temperature between 35° C. and 45° C.In some embodiments, the templating reaction mixture is incubated forbetween 10 and 60 minutes. In some embodiments, at least 100 times asmany substantially monoclonal template nucleic acid molecules arepresent on the templated solid supports as were present on thepre-seeded solid supports.

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses for performing themethods, wherein the methods are for determining the sequences oftemplate nucleic acid molecules, and include: performing a pre-seedingreaction and a subsequent templating reaction. In some embodiments, thepre-seeding reaction generates a plurality of pre-seeded solid supportswherein individual pre-seeded supports in the plurality of pre-seededsolid supports include a plurality of first primers attached to thesolid supports, wherein the plurality of first primers have asubstantially identical sequence, and wherein some of the plurality ofthe first primers are joined to a template nucleic acid molecule andsome of the plurality of the first primers are not joined to a templatenucleic acid molecule. In some embodiments, the disclosure relatesgenerally to methods, as well as systems, compositions, kits andapparatuses for determining the sequences of template nucleic acidmolecules, including: a) generating a population of pre-seeded solidsupports including a population of attached identical first primers,wherein the pre-seeded solid supports are generated under pre-seedingconditions and wherein each of the pre-seeded solid supports has between10 and 100,000 substantially monoclonal template nucleic acid moleculesincluding the first primer attached thereto, and includes attached firstprimers that are attached to the pre-seeded solid supports and are notbound to template nucleic acid molecules; b) forming a templatingreaction mixture by combining the population of pre-seeded solidsupports, nucleotides, a recombinase, a polymerase, and a population ofidentical second primers in solution not attached to any substrate,wherein the template nucleic acid molecules include a primer bindingsite for the second primer at or near the terminal end that is oppositea proximal segment including the first primer; c) initiating atemplating reaction by adding a cation to the templating reactionmixture, wherein template nucleic acid molecules are not present insolution in the reaction mixture when the templating reaction isinitiated; d) incubating the initiated templating reaction mixture underisothermal conditions for at least 10 minutes to amplify the templatemolecules in a templating reaction to generate one or more templatedsolid supports including at least 10 times as many attachedsubstantially monoclonal template nucleic acid molecules on thetemplated solid supports as were present on the pre-seeded solidsupports; and e) sequencing template nucleic acid molecules on the oneor more templated solid supports, thereby determining the sequences oftemplate nucleic acid molecules. In some embodiments, the templatenucleic acid molecules include two or more template nucleic acidmolecules with different sequences. In some embodiments, thesubstantially monoclonal template nucleic acid molecules, which areattached to a pre-seeded solid support and/or are attached to atemplated solid support, include template nucleic acid molecules havingtwo or more different sequences. In further embodiments, thesubstantially monoclonal template nucleic acid molecules attached toeach pre-seeded solid support include at least 70% of all templatenucleic acid molecules attached to each pre-seeded solid support. Insome embodiments, the templated solid supports are templated beads andthe sequencing include distributing the beads in wells of a solidsupport before a sequencing reaction is performed. In some embodiments,each pre-seeded solid support of the population of solid supports hasbetween 10 and 50,000 substantially monoclonal template nucleic acidmolecules attached thereto and the one or more solid supports are beads.In some embodiments, the pre-seeding reaction mixture and/or thetemplating reaction mixture further includes a recombinase-accessoryprotein. In further embodiments, the recombinase-accessory protein is asingle-stranded binding protein and/or a recombinase-loading protein. Insome embodiments, the templating reaction mixture and/or the pre-seedingreaction mixture is incubated at a temperature between 35° C. and 45° C.In some embodiments, the templating reaction mixture is incubated forbetween 10 and 60 minutes. In some embodiments, the pre-seeded solidsupports are generated using a first recombinase-polymeraseamplification (RPA) reaction and the templating reaction is a second RPAreaction. In further embodiments, the first RPA reaction is performed byincubating an RPA reaction mixture for 2 to 5 minutes at a temperaturebetween 35° C. and 45° C. In some embodiments, at least 100 times asmany substantially monoclonal template nucleic acid molecules arepresent on the templated solid supports as were present on thepre-seeded solid supports.

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses for performing themethods, wherein the methods are for determining the sequences oftemplate nucleic acid molecules, and include: a) performing apre-seeding reaction by incubating a first recombinase-polymeraseamplification (RPA) reaction mixture including a population of templatenucleic acid molecules and a population of solid supports including apopulation of attached identical first primers under pre-seedingreaction conditions to generate one or more pre-seeded solid supports,wherein the pre-seeding reaction conditions include incubating the RPAreaction mixture under isothermal conditions and wherein the pre-seededsolid supports each have between 10 and 100,000 substantially monoclonalnucleic acid molecules attached thereto and/or the pre-seeding reactionconditions include incubating the RPA reaction mixture for 2 to 5minutes under isothermal conditions, wherein the pre-seeded solidsupports include: (i.) a substantially monoclonal population of templatenucleic acid molecules attached to the solid support by the firstprimer, and (ii.) attached first primers that are attached to thepre-seeded solid supports and are not bound to template nucleic acidmolecules; b) forming a templating reaction mixture by including the oneor more pre-seeded solid supports in a second RPA reaction mixture,wherein template nucleic acid molecules not associated with thepre-seeded solid supports are not included in the templating reactionmixture, wherein the templating reaction mixture further includes apopulation of identical second primers in solution not attached to anysubstrate, and wherein the template nucleic acid molecules include aprimer binding site for the second primer at or near the terminal endthat is opposite a proximal segment comprising the first primer; c)initiating a templating reaction by adding a cation to the templatingreaction mixture; d) incubating the initiated templating reactionmixture under isothermal conditions for at least 10 minutes to amplifythe template nucleic acid molecules in a templating reaction to generateone or more templated solid supports including at least 10 times as manysubstantially monoclonal template nucleic acid molecules on thetemplated solid supports as were present on the pre-seeded solidsupports; and e) sequencing template nucleic acid molecules on the oneor more templated solid supports, thereby determining the sequence of atemplate nucleic acid molecules. In some embodiments, the templatenucleic acid molecules includes two or more template nucleic acidmolecules with different sequences. In some embodiments, thesubstantially monoclonal template nucleic acid molecules, which areattached to a pre-seeded solid support and/or are attached to atemplated solid support, include template nucleic acid molecules havingtwo or more different sequences. In further embodiments, thesubstantially monoclonal template nucleic acid molecules attached toeach pre-seeded solid support includes at least 70% of all templatenucleic acid molecules attached to each pre-seeded solid support. Insome embodiments, the templated solid supports are templated beads andthe sequencing includes distributing the beads in wells of a solidsupport before a sequencing reaction is performed. In some embodiments,each pre-seeded solid support of the population of solid supports hasbetween 10 and 50,000 substantially monoclonal template nucleic acidmolecules attached thereto and the one or more solid supports are beads.In some embodiments, the pre-seeding reaction mixture and/or thetemplating reaction mixture further includes a recombinase-accessoryprotein. In further embodiments, the recombinase-accessory protein is asingle-stranded binding protein and/or a recombinase-loading protein. Insome embodiments, the templating reaction mixture and/or the pre-seedingreaction mixture is incubated at a temperature between 35° C. and 45° C.In some embodiments, the templating reaction mixture is incubated forbetween 10 and 60 minutes. In some embodiments, at least 100 times asmany substantially monoclonal template nucleic acid molecules arepresent on the templated solid supports as were present on thepre-seeded solid supports.

In some embodiments, the disclosure relates generally to compositions,as well as systems, methods, kits and apparatuses relating to thecompositions, wherein the compositions include a templating reactionmixture including a population of pre-seeded solid supports,nucleotides, a recombinase, and a polymerase, wherein the population ofpre-seeded solid supports have between 10 and 50,000 substantiallymonoclonal template nucleic acid molecules including a first primerattached thereto and further include attached first primers that areattached to the pre-seeded solid support and are not bound to templatenucleic acid molecules, wherein the reaction mixture does not include acation capable of initiating a recombinase-polymerase amplificationreaction, and wherein at least 95% of the template nucleic acidmolecules in the reaction mixture are attached to the one or more solidsupports. In some embodiments, the template nucleic acid moleculesincludes two or more template nucleic acid molecules with differentsequences. In some embodiments, the substantially monoclonal templatenucleic acid molecules, which are attached to a pre-seeded solid supportand/or are attached to a templated solid support, include templatenucleic acid molecules having two or more different sequences. In someembodiments, the pre-seeded solid supports are pre-seeded beads. In someembodiments, the templating reaction mixture includes arecombinase-accessory protein. In further embodiments, therecombinase-accessory protein is a single-stranded binding proteinand/or a recombinase-loading protein. In some embodiments, thepre-seeded solid supports are generated using a firstrecombinase-polymerase amplification (RPA) reaction. In furtherembodiments, the first RPA reaction is performed by incubating an RPAreaction mixture for 2 to 5 minutes at a temperature between 35° C. and45° C. In some embodiments, the pre-seeding reaction mixture furtherincludes a population of identical second primers in solution, and thetemplate nucleic acid molecules includes a primer binding site for thefirst primer at or near a first terminus. In some embodiments, thetemplating reaction mixture further includes a cation capable ofinitiating a recombinase-polymerase amplification reaction.

In some embodiments of the methods for generating one or more templatedsupports, such as, for example, solid supports provided herein, at leastsome, most or all of the nucleic acids attached to the templated supportor supports generated in the method contain at least one modifiednucleotide that includes an attachment, e.g., a first linker moiety,thereto. Such methods may further include linking the templatedsupport(s) to a magnetic bead having a moiety (e.g., a binding partneror second linker moiety) to which the modified nucleotide(s) of thenucleic acids attached to the templated support can bind, link or attachvia the attachment or linker of the modified nucleotide(s) therebyforming a bead assembly of the templated support and the magnetic bead.In some embodiments of these methods, the bead assembly is separatedfrom any elements that do not include a magnetic bead by applying amagnetic field to the bead assembly thereby separating the bead assemblyaway from any such elements and forming an enriched population oftemplated supports. In some embodiments of the methods, the separatedbead assembly is further subjected to conditions under which thetemplated support is released from the magnetic bead and is analyzed infurther methods, e.g., sequencing methods.

Also provided herein are methods of preparing a device for analysis of anucleic acid, such as, for example, sequencing of a nucleic. In someembodiments, the method includes generating a nucleic acid containing acapture sequence portion, a template portion and a primer portioncontaining a first linker moiety, capturing, for example throughhybridization, the nucleic acid on a support, e.g., a solid support suchas a bead, having a plurality of capture primers complementary to thecapture sequence portion of the nucleic acid, linking the first linkermoiety of the captured nucleic acid to a second linker moiety containedon a magnetic bead to form a bead assembly of the captured nucleic acidon the support and the magnetic beads, applying a magnetic field to thebead assembly thereby separating the bead assembly away from anyelements that do not include a magnetic bead, releasing the capturednucleic acid on the support from the magnetic bead, mixing the releasedcaptured nucleic on the support with magnetic beads to which thecaptured nucleic acid on the support does not attach, link or bind andincorporating the mixture into a device for analysis of the capturednucleic acid. In some embodiments, the mixture of captured nucleic acidon the support and magnetic beads is applied to a surface, e.g., a chip,such as a semiconductor chip, and a magnetic field is applied to thesurface. The surface may contain microwells into which the capturednucleic acid on the support is loaded through the movement of themagnetic beads over the surface as the magnetic field is applied to thesurface. In some such embodiments, the size of the magnetic bead is suchthat the magnetic beads cannot enter the microwells.

In some embodiments of the methods of preparing a device for analysis ofa nucleic acid, the method includes generating a nucleic acid containinga capture sequence portion, a template potion and a primer portioncontaining a first linker moiety, capturing the nucleic acid on asupport, e.g., a solid support such as a bead, having a plurality ofcapture primers complementary to the capture sequence portion of thenucleic acid, linking the captured nucleic acid to a magnetic beadhaving a second linker moiety to form a bead assembly such that thefirst and second linker moieties are attached and loading the beadassembly into a well of the device using a magnetic field.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1D are histograms showing the read lengths inhigh-throughput sequencing after bulk isothermal amplification.

FIG. 1A shows the read lengths using Ion Sphere Particles (ISPs) with nopre-seeded template (no template control (NTC)).

FIG. 1B shows the read lengths using ISPs pre-seeded with ˜70copies/ISP.

FIG. 1C shows the read lengths using ISPs pre-seeded with ˜665copies/ISP.

FIG. 1D shows the read lengths using ISPs pre-seeded with ˜4,170copies/ISP (FIG. 1D).

FIGS. 2A-2F are bar graphs showing various metrics of high-throughputsequencing after bulk isothermal amplification using NTC P1 ISPs andISPs pre-seeded with ˜70, ˜665, and ˜4,170 copies/ISP.

FIG. 2A shows the percent ISP loading for NTC ISPs and ISPs pre-seededwith different template copy numbers.

FIG. 2B shows the percent usable reads from NTC ISPs and ISPs pre-seededwith different template copy numbers.

FIG. 2C shows the number of total reads from NTC ISPs and ISPspre-seeded with different template copy numbers.

FIG. 2D shows the mean, median, and mode of the read lengths from NTCISPs and ISPs pre-seeded with different template copy numbers.

FIG. 2E shows the percent empty wells for NTC ISPs and ISPs pre-seededwith different template copy numbers.

FIG. 2F shows the percent low quality wells for NTC ISPs and ISPspre-seeded with different template copy numbers.

FIGS. 3A-3F are bar graphs showing various metrics of high-throughputsequencing after a pre-seeding amplification reaction using ISPs with notemplate control (NTC) and ISPs pre-seeded with ˜82, ˜775, and ˜5,400copies/ISP.

FIG. 3A shows the percent ISP loading for NTC ISPs and ISPs pre-seededwith different template copy numbers.

FIG. 3B shows the percent usable reads from NTC ISPs and ISPs pre-seededwith different template copy numbers.

FIG. 3C shows the number of total reads from NTC ISPs and ISPspre-seeded with different template copy numbers.

FIG. 3D shows the mean, median, and mode of the read lengths from NTCISPs and ISPs pre-seeded with different template copy numbers.

FIG. 3E shows the percentage of empty wells for ISPs pre-seeded withdifferent template copy numbers.

FIG. 3F shows the percentage of low quality wells for ISPs pre-seededwith different template copy numbers.

FIG. 4 is a schematic showing primer extension reactions for the top andbottom strands of a template nucleic acid molecule.

FIG. 5, FIG. 6, and FIG. 7 illustrate example schema for attachingnucleic acids to a bead.

FIG. 8 is an illustration of an example method for preparing asequencing device.

FIG. 9 illustrates example schema for attaching nucleic acids to a bead.

FIG. 10 is a graph of results of sequencing runs of nucleic acidtemplates generated using several different methods.

The above-identified figures are provided by way of representation andnot limitation.

DEFINITIONS

As utilized in this disclosure, the following terms shall be understoodto have the following meanings:

The term “monoclonal” and its variants, when used in reference to one ormore polynucleotide populations, refers to a population ofpolynucleotides where at about 50-99%, or up to 100% of the members ofthe population share at least 80% identity at the nucleotide sequencelevel. As used herein, the phrase “substantially monoclonal” and itsvariants, when used in reference to one or more polynucleotidepopulations, refer to one or more polynucleotide populations wherein anamplified template polynucleotide molecule is the single most prevalentpolynucleotide in the population. Accordingly, all members of amonoclonal or substantially monoclonal population need not be completelyidentical or complementary to each other. For example, differentportions of a polynucleotide template can become amplified or replicatedto produce the members of the resulting monoclonal population;similarly, a certain number of “errors” and/or incomplete extensions mayoccur during amplification of the original template, thereby generatinga monoclonal or substantially monoclonal population whose individualmembers can exhibit sequence variability amongst themselves. In someembodiments, a low or insubstantial level of mixing of non-homologouspolynucleotides may occur during nucleic acid amplification reactions ofthe present teachings, and thus a substantially monoclonal populationmay contain a minority of one or more polynucleotides (e.g., less than50%, less than 40%, less than 30%, less than 20%, less than 10%, lessthan 5%, less than 1%, less than 0.5%, less than 0.1%, or less than0.001%, of diverse polynucleotides). In certain examples, at least 90%of the polynucleotides in the population are at least 90% identical tothe original single template used as a basis for amplification toproduce the substantially monoclonal population. In certain embodiments,methods for amplifying yield a population of polynucleotides wherein atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% of the members of a population of polynucleotidesshare at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the template nucleic acid from whichthe population was generated. In certain embodiments, methods foramplifying yield a population of polynucleotides in which a large enoughfraction of the polynucleotides share enough sequence identity to allowsequencing of at least a portion of the amplified template using ahigh-throughput sequencing system.

In some embodiments, at least 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%,of the members of the template nucleic acid molecules attached totemplated supports will share greater than 90%, 95%, 97%, 99%, or 100%identity with the template nucleic acid molecule. In some embodiments,members of a nucleic acid population which are produced using any of theamplification methods, hybridize to each other under high-stringencyhybridization conditions.

In some embodiments, the amplification methods generate a population ofsubstantially monoclonal nucleic acid molecules that includessufficiently few polyclonal contaminants such that they are successfullysequenced in a high-throughput sequencing method. For example, theamplification methods can generate a population of substantiallymonoclonal nucleic acid molecules that produces a signal (e.g., asequencing signal, a nucleotide incorporation signal and the like) thatis detected using a particular sequencing system. Optionally, the signalcan subsequently be analyzed to correctly determine the sequence and/orbase identity of any one or more nucleotides present within any nucleicacid molecule of the population. Examples of suitable sequencing systemsfor detection and/or analysis of such signals include the Ion Torrentsequencing systems, such as the Ion Torrent PGM™ sequence systems,including the 314, 316 and 318 systems, the Ion Torrent Proton™sequencing systems, including Proton I, (Thermo Fisher Scientific,Waltham, Mass.) and the Ion Torrent Proton™ sequencing systems,including Ion S5 and S5XL (Thermo Fisher Scientific, Waltham, Mass.). Insome embodiments, the monoclonal amplicon permits the accuratesequencing of at least 5 contiguous nucleotide residues on an IonTorrent sequencing system.

As used herein, the term “clonal amplification” and its variants referto any process whereby a substantially monoclonal polynucleotidepopulation is produced via amplification of a polynucleotide template.In some embodiments of clonal amplification, two or more polynucleotidetemplates are amplified to produce at least two substantially monoclonalpolynucleotide populations.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Other features and advantages of the present disclosure will be apparentfrom the following detailed description and from the claims.

DETAILED DESCRIPTION

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses for performing themethods, of manipulating nucleic acids. In some embodiments, the methodsinclude nucleotide polymerization, nucleotide sequence modification(e.g., ligation of adapter and/or primer sequences, such as, forexample, cleavable primers), nucleotide modification (e.g., addition oflabels or linker molecules (e.g., biotin) to a one or more nucleotidesin a nucleic acid molecule), nucleic acid amplification, nucleic acidcapture, nucleic acid containment and/or nucleic acid transfer. Methodsand compositions provided herein can be used, for example, to generate amonoclonal, or substantially monoclonal, population of nucleic acids. Insome embodiments, the methods are used to generate a monoclonal, orsubstantially monoclonal, population of nucleic acids in which thenucleic acids are attached to a support, such as, for example, a solidsupport. In some embodiments, the methods are used to attach a nucleicacid to two or more supports. In such embodiments, the two or moresupports can be the same or different, including, for example, polymericand/or magnetic supports. In some embodiments, the methods are used totransfer a nucleic acid attached to one or more supports to a reactionchamber. In some embodiments, the methods are used to generate amonoclonal, or substantially monoclonal, population of nucleic acidswithin a reaction chamber or multiple reaction chambers. The methodsprovided herein can be performed alone or in any combination for any oneor more uses and provide advantages in the analysis of nucleic acids.For example, use of methods and/or compositions disclosed herein providefor improved quality, quantity and/or accuracy of nucleic acid analysisresults, such as, for example, nucleic acid sequencing results. Inanother example, use of methods and/or compositions disclosed hereinalone and/or in any combination enhance nucleic acid manipulationprocesses to markedly facilitate workflow automation of nucleic acidanalysis, such as, for example, nucleic acid sequencing.

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses, that improvehigh-throughput nucleic acid sequencing results. The methods, systems,compositions, kits and apparatuses incorporate processes andcompositions for generating, containing, isolating, transferring,replicating and/or manipulating substantially monoclonal populations ofnucleic acids that are rapid, efficient and cost-effective whileproviding for significantly increased production of high-quality readsof longer length with decreased numbers of duplicate, noninformativeand/or bank reads and run times compared to existing methods. In someembodiments, the methods, as well as systems, compositions, kits andapparatuses include supports, e.g., solid supports, to confine, enrich,sequester, isolate, localize, amplify and/or transfer nucleic acids thatcan be used in analysis methods. In some embodiments, the supports arepre-seeded with one or a limited number of predominantly substantiallyidentical nucleic acids from a large collection of nucleic acids (e.g.,a nucleic acid library or sample) to provide an isolated single nucleicacid molecule or localized substantially monoclonal population. Suchpre-seeded supports are readily manipulated and used as clean source ofidentical nucleic acids that can be clonaly amplified, for example, intemplating reactions, to generate a relatively pure collection oftemplates for use in a high-throughput sequencing workflow to improvesequencing results. In some embodiments, the pre-seeded supports can begenerated, at least in part, and expanded using isothermalamplification, especially recombinase-mediated amplification reactionssuch as recombinase-polymerase amplification (RPA). Accordingly, inillustrative aspects of the methods, a pre-seeding reaction is carriedout before a templating reaction in a high-throughput sequencingworkflow.

In a typical high-throughput sequencing workflow, nucleic acid moleculesin a sample are used to prepare a library of template nucleic acidmolecules suitable for downstream sequencing. Multiplex amplificationcan optionally be performed on the nucleic acid molecules before,during, or after library preparation. The library of template nucleicacid molecules is then amplified onto one or more supports to be used ina sequencing reaction. In some embodiments of the present disclosure,the amplification of a template on a solid support typically isperformed in two or more reactions, including, for example, one or morepre-seeding reactions that generates one or more pre-seeded supportswith substantially monoclonal populations of the template nucleic acidmolecules attached, followed by a templating reaction on the pre-seededsupports that generates more copies (e.g., at least 10× more copies) ofthe attached template nucleic acid molecules on the one or moresupports. The advantage of performing a pre-seeding reaction and atemplating reaction is that this workflow generates more high-qualitysequencing reads in a high-throughput sequencing reaction. In someembodiments, the pre-seeding reaction is performed with blocked primersto prevent the formation of primer dimer amplicons during thepre-seeding reaction. In previous methods, primer dimer amplicons couldbe generated during templating, which could generate lower qualitysequencing reads and a reduction in the quantity of sequencing readsfrom the template nucleic acid molecules. Therefore, the addition of aseparate pre-seeding reaction provides this and significant otheradvantages over prior methods.

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses for amplifying atemplate nucleic acid molecule that include a templating reactionmixture using pre-seeded supports. In various aspects, the templatedsupports are used for downstream sequencing methods. The templatingreaction typically uses a recombinase to denature double-strandedtemplate nucleic acid molecules and is carried out at isothermaltemperatures with a suitable polymerase for an RPA reaction. In someaspects, the pre-seeded supports are generated in a separate pre-seedingreaction mixture.

Pre-seeding, also referred to herein as seeding, involves the attachmentof one or more nucleic acid molecules to a support. In some embodiments,the attachment is designed to generate a single support with a singlenucleic acid molecule attached thereto. In some embodiments, theattachment is designed to attach multiple copies of a nucleic acidmolecule thereto and/or multiple different nucleic acids thereto. Insome embodiments, the attachment is designed to attach a limited numberof multiple copies of substantially the same nucleic acid thereto togenerate a substantially monoclonal population of nucleic acids.

In some embodiments, a pre-seeding (or seeding) method provided hereinincludes hybridizing a single-stranded nucleic acid molecule to acomplementary nucleic acid, e.g., an oligonucleotide, that is bound toand immobilized on the support. Such methods carried out under annealingconditions over a typically short time period. In some embodiments, theseeding method involves hybridizing under conditions in which the solidsupport, e.g., a bead, will have only one single-stranded nucleic acidmolecule attached to it. Such conditions include contacting a populationof single-stranded nucleic acids (e.g., from a library or sample ofnucleic acids) with a substantial excess of supports under annealingconditions.

In some embodiments, a pre-seeding (or seeding) method provided hereinincludes a combined process of attaching a nucleic acid to a supportthrough hybridization and, at the same time, amplifying the attachednucleic acid to a low level, for example, about 20 copies. In theseembodiments, the pre-seeding reaction mixture typically includes apopulation of template nucleic acid molecules, a polymerase,nucleotides, a population of first primers, and a cofactor such as adivalent cation. A skilled artisan will understand that a variety ofmethods can be used to pre-seed solid supports with substantiallymonoclonal template nucleic acid molecules. As non-limiting examples,the pre-seeding reaction mixture can be carried out using an RPAreaction, a template walking reaction, PCR, emulsion PCR, or bridge PCR.The pre-seeding reaction and/or the templating reaction can be performedin bulk in a solution. Furthermore, the pre-seeding reaction mixtureand/or the templating reaction mixture can include a first universalprimer attached to one or more supports, a second universal primer insolution (a soluble second universal primer), and a plurality oftemplate nucleic acid molecules where individual template nucleic acidmolecule are joined to at least one universal primer binding sequence(s)which may be added during library preparation, and where the universalprimer binding sequence(s) bind the first and optional second universalprimer(s). In some embodiments, the pre-seeding reaction and/or thetemplating reaction is performed in wells. In illustrative examples, thepre-seeding reaction and the templating reaction are carried out usingconsecutive RPA reactions, wherein template nucleic acid molecules arewashed away after the pre-seeding reaction before performing thetemplating reaction.

Loading supports, e.g., beads, modified with nucleic acid molecules intoconfined regions or receptacles, such as microwells or dimples, to forman array presents several advantages for nucleic acid sequencing.Placing nucleic acid-coated beads in an organized, tightly packedfashion, for example, into small microwells, can increase throughput percycle and lower customer cost. As the density of microwells increases oras the microwell size decreases, bead loading becomes difficult, leadingto many open microwells and low counts of beads in wells. Too many openmicrowells provides for a decreased number of base reads and thus, poorsequencing performance. Provided herein, in some embodiments, aremethods, as well as systems, compositions, kits and apparatuses for usein the methods, of introducing a support, such as a solid support, e.g.,a bead, into a microwell or reaction chamber. In some embodiments, themethod includes linking a bead support having a captured templatenucleic acid modified with a linker moiety to a magnetic bead havingcomplementary linker moiety to form a bead assembly and loading the beadassembly into a well using a magnetic field. The bead assembly can bedenatured to release the magnetic bead, leaving the bead supportattached to a target nucleic acid in the well. Such methods can be used,for example, in preparing a device for sequencing of nucleic acids. Inan embodiment, reactions carried out in the well can be analyticalreactions to identify or determine characteristics or properties of ananalyte of interest. Such reactions can generate directly or indirectlybyproducts that provide a signal which can indicate if the analytereacts in a characteristic manner. If such byproducts are produced insmall amounts or rapidly decay or react with other constituents, thenmultiple copies of the same analyte may be analyzed in the well at thesame time in order to increase the output signal generated. In anembodiment of methods provided herein, multiple copies of an analyte,e.g., a nucleic acid, may be attached to a solid phase support, eitherbefore or after deposition into the well. For example, a target nucleicacid can be amplified in the microwell to provide a clonal population oftarget nucleic acids useful for sequencing the target nucleic acid. Thesolid phase support may be, for example, microparticles, nanoparticles,beads, solid or porous comprising gels, or the like. For simplicity andease of explanation, solid phase supports are also referred to herein asa particle or bead. For a nucleic acid analyte, multiple, connectedcopies may be made by rolling circle amplification (RCA), exponentialRCA, or like techniques, to produce an amplicon without the need of asolid support.

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses for generating oneor more templated solid supports, including: a) forming a templatingreaction mixture by combining one or more pre-seeded solid supports,nucleotides, a recombinase, and a polymerase, wherein the one or morepre-seeded solid supports include a population of attached substantiallyidentical first primers and have substantially monoclonal templatenucleic acid molecules attached thereto, wherein the substantiallymonoclonal template nucleic acid molecules include a proximal segmentincluding the first primer, wherein the proximal segment attaches atemplate nucleic acid segment to a solid support at the first primer andwherein the pre-seeded solid supports further include attached firstprimers that are attached to the pre-seeded solid support and are notbound to template nucleic acid molecules, wherein the templatingreaction mixture further includes a population of substantiallyidentical soluble second primers, and wherein the template nucleic acidmolecules include a primer binding site for the second primer at or nearthe terminus opposite the proximal segment; and b) performing atemplating reaction by adding a cation to the templating reactionmixture and incubating the reaction mixture under isothermal conditionsfor at least 10 minutes to amplify the template nucleic acid moleculesin a templating reaction to generate one or more templated solidsupports. In some embodiments, the method provides templated solidsupports wherein each support includes at least 100,000 substantiallymonoclonal template nucleic acid molecules. Typically, template nucleicacid molecules are not included in solution in the templating reactionmixture. As such, template nucleic acid molecules are typically presentin solution in the reaction mixture when the templating reaction isinitiated. The pre-seeded solid supports are typically generated in apre-seeding reaction that is separate from the templating reaction. Insome embodiments, template nucleic acids are attached to the solidsupport by a proximal segment that does not include 100 or moreidentical nucleotides. In some embodiments, the proximal segment doesnot include a contiguous sequence of more than 2, 3, 4, 5, 6, 7, 8, 9 or10 identical nucleotides. In some embodiments of the above aspect, theone or more templated solid supports are used in a sequencing reactionto determine the sequences of the template nucleic acid molecules.

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses, for determining thesequences of template nucleic acid molecules, including: a) generating apopulation of pre-seeded solid supports including a population ofattached substantially identical first primers, wherein the pre-seededsolid supports are generated under pre-seeding conditions and whereineach of the pre-seeded solid supports has between 10 and 100,000substantially monoclonal template nucleic acid molecules including thefirst primer attached thereto, and further includes attached firstprimers that are attached to the pre-seeded solid supports and are notbound to template nucleic acid molecules; b) forming a templatingreaction mixture by combining the population of pre-seeded solidsupports, nucleotides, a recombinase, and a polymerase; c) initiating atemplating reaction by adding a cation to the templating reactionmixture, wherein template nucleic acid molecules are not present insolution in the reaction mixture when the templating reaction isinitiated; d) incubating the initiated templating reaction mixture underisothermal conditions for at least 10 minutes to amplify the templatemolecules in a templating reaction to generate one or more templatedsolid supports including at least 10 times as many attachedsubstantially monoclonal template nucleic acid molecules on thetemplated solid supports as were present on the pre-seeded solidsupports; and d) sequencing template nucleic acid molecules on the oneor more templated solid supports, thereby determining the sequences oftemplate nucleic acid molecules.

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses, for determining thesequences of template nucleic acid molecules, including: a) performing apre-seeding reaction by incubating a recombinase-polymeraseamplification (RPA) reaction mixture including a population of templatenucleic acid molecules and a population of solid supports including apopulation of attached substantially identical first primers underpre-seeding reaction conditions to generate one or more pre-seeded solidsupports including substantially monoclonal population of templatenucleic acid molecules attached to the solid support by the first primerand attached first primers that are attached to the pre-seeded solidsupports and are not bound to template nucleic acid molecules, whereinthe pre-seeding reaction conditions include incubating the RPA reactionmixture under isothermal conditions, wherein the pre-seeded solidsupports each have between 10 and 100,000 substantially monoclonalnucleic acid molecules attached thereto and/or the pre-seeding reactionconditions include incubating the RPA reaction mixture for 2 to 5minutes under isothermal conditions; b) forming a templating reactionmixture by including the one or more pre-seeded solid supports in an RPAreaction mixture, wherein template nucleic acid molecules not associatedwith the pre-seeded solid supports are not included in the templatingreaction mixture; c) initiating a templating reaction by adding a cationto the templating reaction mixture; d) incubating the initiatedtemplating reaction mixture under isothermal conditions for at least 10minutes to amplify the template nucleic acid molecules in a templatingreaction to generate one or more templated solid supports including atleast 10 times as many substantially monoclonal template nucleic acidmolecules on the templated solid supports as were present on thepre-seeded solid supports; and e) sequencing the template nucleic acidmolecules on the one or more templated solid supports, therebydetermining the sequence of a template nucleic acid molecule.

In some embodiments, the plurality of template nucleic acid moleculesincludes two or more template nucleic acid molecules with differentsequences. In some embodiments, the substantially monoclonal templatenucleic acid molecules, which are attached to a pre-seeded solid supportand/or are attached to a templated solid support, include templatenucleic acid molecules having two or more different sequences. Thesubstantially monoclonal template nucleic acid molecules typically areattached to a solid support by a primer that can include consecutiveidentical nucleotides, or no consecutive identical nucleotides or nomore than 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive nucleotides or by aprimer that includes consecutive non-identical nucleotides. However, theproximal segment of a template nucleic acid which is the segmentattached to a solid support and is typically a primer, includes fewerthan 100 identical nucleotides. In some embodiments, the templated solidsupports are templated beads and the sequencing includes distributingthe beads in wells of a solid support before a sequencing reaction isperformed. Each pre-seeded solid support of the population of solidsupports, in illustrative examples, can have, for example, between 10and 50,000 or between 100 and 25,000 substantially monoclonal templatenucleic acid molecules attached thereto. In illustrative example, thepre-seeding reaction mixture and/or the templating reaction mixturefurther include a population of identical second primers in solution. Inthese examples, the template nucleic acid molecules typically include aprimer binding site for the first primer at or near a first terminus anda primer bind site for the second primer at or near the other terminus.

In another aspect, the templating reaction mixture including apopulation of pre-seeded solid supports, nucleotides, a recombinase, anda polymerase, wherein the population of pre-seeded solid supports havebetween 10 and 50,000 substantially monoclonal template nucleic acidmolecules including a first primer attached thereto and further includeattached first primers not bound to template nucleic acid molecules,wherein the reaction mixture does not include a cation capable ofinitiating a recombinase-polymerase amplification reaction, and whereinat least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% of the templatenucleic acid molecules in the reaction mixture are attached to the oneor more solid supports.

In some embodiments, the one or more pre-seeded supports are formedduring a pre-seeding reaction using a pre-seeding reaction mixture. Insome embodiments, the pre-seeding reaction mixture includes some or allof the following: a population of template nucleic acid molecules, oneor more supports, a polymerase, a population of first primers,nucleotides, and a divalent cation. In some embodiments, the populationof first primers is attached to the one or more supports. In any of theembodiments of the present teachings, the pre-seeding reaction mixturefurther includes a second primer and optionally a diffusion-limitingagent. In illustrative embodiments, the second primer is in solution. Inillustrative embodiments, the pre-seeding reaction mixture includes arecombinase and optionally a recombinase accessory protein.

In some embodiments, the pre-seeding and/or the templating reaction(s)are conducted by employing nucleic acid amplification using (i) aplurality of nucleic acid molecules which include a target sequence andone or more universal adaptor sequences, (ii) a plurality of solubleforward primers, (iii) a plurality of soluble reverse blocked tailedprimers, and (iv) a plurality of solid supports having immobilizedthereon capture primers that hybridize to a universal adaptor sequence.In some embodiments, the pre-seeding and/or templating reactions areconducted in a single reaction mixture with a plurality of nucleic acidmolecules having the same or different target sequences. In someembodiments, individual nucleic acid molecules generated from a sampleinclude a target sequence joined at the ends to at least one universaladaptor sequence (e.g., an A-adaptor and/or P1-adaptor sequences). Insome embodiments, the nucleic acid molecules are double-strandedmolecules having complementary top and bottom strands. In someembodiments, the pre-seeding reaction is conducted to amplify and attachsubstantially monoclonal copies of a nucleic acid molecule to a solidsupport using forward and reverse soluble primers that hybridize to theadaptor sequences (see FIG. 4). Although FIG. 4 depicts a series ofreactions for a single double-stranded nucleic acid molecule and asingle bead within a single reaction mixture, it will be appreciated bythe skilled artisan that the same single reaction mixture contains aplurality of double-stranded nucleic acid molecules and a plurality ofbeads that are undergoing the same series of reactions to generate atleast two beads each attached with a substantially monoclonal populationof target sequences. Additionally, the skilled artisan will appreciatethat the bead can be attached a plurality of B capture primers. Further,the bottom of FIG. 4 depicts a biotinylated primer extension productthat binds to a B capture primer, but it will be appreciated by theskilled artisan that a non-biotinylated primer extension product canbind a B capture primer. It will also be appreciated by the skilledartisan that a mixture of biotinylated and non-biotinylated primerextension products can be attached to the plurality of B capture primersthat are attached to the bead. In some embodiments, the single reactionmixture contains a plurality of nucleic acid molecules, whereinindividual nucleic acid molecules having the same or different targetsequences are attached to at least one universal adaptor sequence.Referring now to FIG. 4, the nucleic acid molecules, having top andbottom strands, is denatured and the separated top and bottom strands isused in primer extension reactions using soluble primers (e.g., solubleA primers) and soluble blocked tailed primers (e.g., soluble blockedtailed P1/B primers), to generate a plurality of primer extensionproducts having adapter sequences that can bind an immobilized B primerduring the pre-seeding and/or templating reactions. As shown in FIG. 4,the primer extension reactions generate different products for the twostrands due to the different sequences and orientations of the top andbottom strands and differences in the primers used.

In some embodiments, in a first primer extension reaction, acomplementary strand of the bottom strand is generated using a solubleprimer that binds the A primer binding site. For illustration purposesin FIG. 4 (left) the soluble A′ primer is complementary to the A adaptorsequence. In some embodiments, a mixture of varying length soluble A′primers is used for the first primer extension reaction. The mixture ofsoluble A′ primers can vary in length at their 5′ ends, 3′ ends, or both5′ and 3′ ends. For example, Primer Mix S (a mix of A′ primers that caninclude various lengths of a 5′ non-complementary sequence with orwithout a 5′ biotin adduct (depicted as “Bio” in FIG. 4), and can beused in the primer extension reaction to generate one of severalpossible first extension products depending on which soluble A′ primeris used for the first primer extension reaction (FIG. 4, left). Forexample, the first extension products contain, in a 5′ to 3′ direction,a complementary A-adaptor sequence (shown as A′ in FIG. 4, left), acomplementary bottom sequence (shown as bottom′ in FIG. 4, left), and acomplementary P1 sequence (shown as P1′ in FIG. 4, left). In someembodiments, in a second primer extension reaction, the newlysynthesized P1′ sequence in the first extension product can bind to asoluble P1 primer to allow primer extension to occur from the 3′ end ofthe P1′ sequence of the first primer extension product. The soluble P1primer can be a tailed primer. The soluble P1 primer can carry ablocking moiety at its 3′ end, wherein the blocking moiety can inhibitprimer extension from the 3′ end of the primer. The soluble P1 primercan be a reverse tailed P1 primer which includes an attached 5′ Badapter sequence such that primer extension of the first extensionproduct, using the tailed primer P1 as a template results in theaddition of the complement of the B sequence (depicted as B′ in FIG. 4left) to the 3′ end. In illustrative embodiments, the soluble P1 primercan have a 3′ blocked end (shown as an encircled “X” in FIG. 4, left) toprevent extension from the 3′ end of the soluble P1 primer (FIG. 4,left). The second primer extension reaction can generate a plurality ofsecond extension products having various lengths, depending on whichsoluble A′ primer was used in the first extension reaction. Theplurality of second extension products contain, in a 5′ to 3′ direction,a complementary A-adaptor sequence (shown as A′ in FIG. 4, left), acomplementary bottom strand sequence (shown as bottom′ in FIG. 4, left),a complementary P1 sequence (shown as P1′ in FIG. 4, left), and acomplementary B adaptor sequence (shown as B′ in FIG. 4, left). Thesecond primer extension products can include or lack a 5′ biotin adduct(FIG. 4, left). The second extension reaction can generate a pluralityof second extension products having different lengths, and which caninclude or lack a biotin adduct, and which include a B′ adaptorsequence. Any of these second extension products can bind/hybridize tothe B capture sequence which is immobilized to the solid surface (bead).The immobilized B primer can undergo a third primer extension reaction,thereby generating a third extension product which is immobilized to thebead, and is complementary to the second extension product (FIG. 4,bottom).

Referring now to FIG. 4 (right) which depicts the double-strandednucleic acid undergoing denaturation, and a series of reactions for thetop strand. In some embodiments, the P1′ adaptor sequence of the topstrand can bind the soluble P1 primer and undergo a first primerextension reaction to generate a first extension product (FIG. 4,right). In some embodiments, the soluble P1 primer is a tailed primer.The soluble P1 primer can carry a blocking moiety at its 3′ end, whereinthe blocking moiety can inhibit primer extension from the 3′ end of theprimer (FIG. 4, right). The soluble P1 primer can be a tailed P1 primerwhich includes an attached 5′ B adapter sequence such that primerextension, using the tailed primer P1 as a template, results in theaddition of the complement of the B sequence (B′) to the 3′ end of thefirst extension product (FIG. 4, right). In illustrative embodiments,the soluble P1 primer can have a 3′ blocked end (shown as an encircled“X” in FIG. 4, right) to prevent extension from the 3′ end of the P1primer (FIG. 4, right). The first primer extension reaction generates aplurality of first extension products which contain, in a 5′ to 3′direction, an A′ adaptor sequence, a top strand sequence, a P1′ adaptorsequence, and a B′ adaptor sequence (FIG. 4, right). The first extensionproduct, which includes a B′ adaptor sequence, can bind/hybridize to theB capture sequence which is immobilized to the solid surface (bead). Theimmobilized B primer can undergo a second primer extension reaction,thereby generating a second extension product which is immobilized tothe bead, and is complementary to the first extension product (FIG. 4,bottom).

In some embodiments, a pre-seeding (or seeding) reaction can beperformed as illustrated in FIG. 5. In this example, a targetpolynucleotide B-A′ and its complement, a template polynucleotide(A-B′), are amplified in the presence of a bead support having a captureprimer. The target polynucleotide has a capture portion (B) the same asor substantially similar to a sequence of the capture primer coupled tothe bead support. Substantially similar sequences are sequences whosecomplements can hybridize to each of the substantially similarsequences. The bead support can have a capture primer that is the samesequence or a sequence substantially similar to that of the B portion ofthe target polynucleotide to permit hybridization of the complement ofthe capture portion (B) of the target polynucleotide with the captureprimer attached to the bead support. Optionally, the targetpolynucleotide can include a second primer location (P1) adjacent to thecapture portion (B) of the target polynucleotide and can further includea target region adjacent the primers and bounded by complement portion(A′) to a sequencing primer portion (A) of the target polynucleotide.When amplified in the presence of the bead support including a captureprimer, the template polynucleotide complementary to the targetpolynucleotide can hybridize with the capture primer (B). The targetpolynucleotide can remain in solution. The system can undergo anextension in which the capture primer B is extended complementary to thetemplate polynucleotide yielding a target sequence bound to the beadsupport. A further amplification can be performed in the presence of afree primer (B), the bead support, and a free modified sequencing primer(A) a having a linker moiety (L) attached thereto. The primer (B) andthe modified primer (L-A) can interfere with the free floating targetpolynucleotide and template polynucleotide, hindering them from bindingto the bead support and each other. In particular, the modifiedsequencing primer (A) having the linker moiety attached thereto canhybridize with the complementary portion (A′) of the targetpolynucleotide attached to the bead support. Optionally, the linkermodified sequencing primer L-A hybridized to the target polynucleotidecan be extended forming a linker modified template polynucleotide. Suchlinker modified template polynucleotide hybridize to the target nucleicacid attached to the bead support can then be captured by a magneticbead and used for magnetic loading of the sequencing device. Theamplification or extensions can be performed using polymerase chainreaction (PCR) amplification, recombinase polymerase amplification(RPA), or other amplification techniques. In a particular example, eachstep of the scheme illustrated in FIG. 5 is performed using PCRamplification.

In some embodiments, a pre-seeding (or seeding) reaction can beperformed as illustrated in FIG. 6. In this example, an alternativescheme includes a target polynucleotide (P1-A′) and its complementtemplate polynucleotide (A-P1′). The target polynucleotide and templatepolynucleotide are amplified in a solution including a linker modifiedsequencing primer (L-A) and a truncated P1 primer (trP1) having aportion having the sequence of the capture primer (B). In an example,the truncated P1 primer (trP1) includes a subset of the sequence of P1or all of the sequence P1. During subsequent amplifications in thepresence of the linker modified sequencing primer (L-A) and truncated P1primer (trP1-B), a single species 702 includes a linker modifiedtemplate polynucleotide (L-A-B′) operable to hybridize with a beadsupport having a capture primer (B). Accordingly, the linker modifiedtemplate polynucleotide (L-A-B′) hybridizes with the capture primer (B)on the bead and is extended to form a target polynucleotide (B-A′)attached to the bead support. The linker modified templatepolynucleotide hybridized to the target polynucleotide attached bead canbe utilized to attach to a magnetic bead, which, for example, can beused to implement magnetic loading of the bead into a sequencing deviceand/or for enriching the nucleic acids attached to the bead. The linkermoiety of the linker modified template polynucleotide can take variousforms, such as biotin, which can bind to linker moieties attached to themagnetic bead, such as streptavidin. Each of the amplification reactionscan be undertaken using PCR, RPA, or other amplification techniques. Inthe example illustrated in FIG. 6, the scheme can be implemented usingthree cycles of polymerase chain reaction (PCR). Such a series of PCRreactions results in a greater percentage of bead supports having asingle target polynucleotide attached thereto. As a result, moremonoclonal populations can be generated in wells in the sequencingdevice.

In some embodiments, a pre-seeding (or seeding) reaction can beperformed as illustrated in FIG. 7. In this example, the reaction isdesigned to generate a desired bead-attached nucleic acid molecule froma series of amplification cycles in which only one of the amplificationproducts, which is the desired target nucleic acid, will attach to thebead. The desired target contains a linker moiety, e.g., biotin,(labeled with the letter “L” in FIG. 7) attached to the 5′ end of thenucleic acid and an adapter nucleotide sequence (labeled with the letter“B′” in FIG. 7) at the 3′ end that is complementary to the primer(labeled with the letter “B” in FIG. 7) immobilized on the bead. Incontrast, the method shown in FIG. 6 generates two nucleic acidamplification products that hybridize to the bead, only one of which hasthe desired linker moiety. In generating only one amplification productthat will attach to the bead, this method avoids the production of beadsthat do not contain the desired target nucleic acid, for example, onelacking linker moiety, which would not be used in downstream analyses.Thus, this method avoids waste of beads and nucleic acids and ensuresthat only a single nucleic acid target molecule will hybridize to a beadwhich is desirable to maintain a high level of monoclonality insubsequent templating amplifications using the beads that have only onenucleic acid template bound thereto. As shown in FIG. 7, thedouble-stranded library nucleic acids contain an A adapter sequence atthe 5′ end and a P1 adapter sequence at the 3′ end (standard Ion TorrentA and P1 library adapters; Thermo Fisher Scientific). The primers usedin the amplification are a biotinylated primer A (forward primer) andreverse primer that is a fusion of trP1 and B primers (trP1 is a 23mersegment of the Ion P1 adapter with sequence CCT CTC TAT GGG CAG TCG GTGAT; SEQ ID NO: 1). The B primer sequence is identical to the sequence ofthe B primer immobilized on the bead. The trP1-B fusion primer willhybridize and prime at the inner portion of the P1 adapter sequence ofthe library nucleic acid molecules, close to the library insert sequenceand does not hybridize at with the remainder of the P1 adapter sequenceat the extreme 3′ end of the library nucleic acids. This forms amismatch end between the trP1-B primer sequence and the very 3′ endportion of the P1 adapter on the library nucleic acids. As shown in FIG.7, after two cycles of amplification (e.g., PCR), although fouramplification products are generated, only one product will be able toseed (or hybridize) to the bead (e.g., an Ion Sphere Particle). Thus,upon subsequent denaturation of the amplification products, asingle-strand of only one of the products will hybridize to the B primeron the bead. This primer can be extended to form a double-strandedtemplate nucleic acid in which one strand contains a linker moiety thatcan be used, for example, to bind the bead-bound nucleic acid to amagnetic bead for use in enrichment and/or magnetic loading of wells.

In some embodiments, after conducting the pre-seeding and/or templatingreactions using the soluble blocked tailed P1/B primers (e.g., asdepicted in FIG. 4), streptavidin beads are used to enrich beads whichare attached with target nucleic acid molecules carrying a biotinadduct. In some embodiments, the beads (enriched or not) that areattached to target nucleic acids and generated according to the seriesof reactions depicted in FIG. 4 are sequenced, for example in amassively parallel sequencing reaction. In some embodiments, the beads(enriched or not) that are attached with target nucleic acid moleculesare deposited on an array of reaction chambers which are coupled tofield effect transistors (FET) or ion-sensitive field effect sensors(ISFE), and the target nucleic acid molecules are sequenced.

In some embodiments, the template nucleic acid molecules are derivedfrom a sample that is from a natural or non-natural source. In someembodiments, the nucleic acid molecules in the sample are derived from aliving organism or a cell. Any nucleic acid molecule can be used, forexample, the sample can include genomic DNA covering a portion of or anentire genome, mRNA, or miRNA from the living organism or cell. In otherembodiments, the template nucleic acid molecules are synthetic orrecombinant. In some embodiments, the sample contains nucleic acidmolecules having substantially identical sequences or having a mixtureof different sequences. Illustrative embodiments are typically performedusing nucleic acid molecules that were generated within and by a livingcell. Such nucleic acid molecules are typically isolated directly from anatural source such as a cell or a bodily fluid without any in vitroamplification. Accordingly, the sample nucleic acid molecules are useddirectly in subsequent steps. In some embodiments, the nucleic acidmolecules in the sample can include two or more nucleic acid moleculeswith different sequences.

A variety of methods are known in the art to prepare template nucleicacid molecules from a sample and can be used in any of the aspects ofthe pre-seeding and/or templating methods, as well as systems,compositions, kits and/or apparatuses. In some embodiments, the nucleicacid molecules are present in the sample as fragments or unfragmented.In any of the disclosed embodiments, the nucleic acid molecules in thesample are fragmented or further fragmented to generate nucleic acidmolecules of any chosen length before being used in the pre-seedingreaction. A skilled artisan will recognize methods for performing suchfragmentation to obtain fragments within a range of chosen lengths. Forexample, the nucleic acid molecules can be fragmented using physicalmethods such as sonication, enzymatic methods such as digestion by DNaseI or restriction endonucleases, or chemical methods such as applyingheat in the presence of a divalent metal cation. In some embodiments,the nucleic acid molecules in the sample are fragmented to generatenucleic acid molecules of any chosen length. A skilled artisan willrecognize methods for performing such fragmentation to achieve a rangeof chosen lengths. In other embodiments, nucleic acid fragments within arange of chosen lengths are selected using methods known in the art. Insome aspects, nucleic acid molecules or nucleic acid fragments areselected for specific size ranges using methods known in the art. Insome embodiments, the nucleic acid molecules or fragments are betweenabout 2 and 10,000 nucleotides in length, for example between about 2and 5,000 nucleotides, between about 2 and 3,000 nucleotides, or betweenabout 2 and 2,000 nucleotides in length.

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses, the nucleic acidmolecules from the sample are used as part of a high-throughputsequencing workflow, such as to generate a library of template nucleicacid molecules. In some embodiments, a population of different templatenucleic acid molecules amplified using any of the amplification methodsof the present teachings include a library of template nucleic acidmolecules having a nucleic acid adapter sequence on one or both ends.For example, the template nucleic acid molecules in the library caninclude a first and second end, where the first end is joined to a firstnucleic acid adapter. The template nucleic acid molecules in the librarycan also include a second end joined to a second nucleic acid adapter.The first and second nucleic acid adapters can be ligated or otherwiseintroduced to the template nucleic acid molecules. The adapter can beligated or otherwise introduced to an end of a linear template, orwithin the body of a linear or circular template nucleic acid molecule.Optionally, the template nucleic acid molecule can be circularized afterthe adapter is ligated or introduced. In some embodiments, a firstadapter is ligated or introduced at a first end of a linear template anda second adapter is ligated or introduced at a second end of thetemplate. The first and second adapters can have the same or differentsequences. The first and second adapters can have primer bindingsequences that are the same or different. In some embodiments, at leasta portion of the first or second nucleic acid adapter (i.e., as part ofthe template nucleic acid molecules in the library) can hybridize to thefirst primer, which is a universal primer.

The nucleic acid molecules can have 5′ and/or 3′ overhangs that can berepaired before further library preparation. In illustrativeembodiments, the template nucleic acid molecules with 5′ and 3′overhangs are repaired to generate blunt-ended sample nucleic acidmolecules using methods known in the art. For example, in an appropriatebuffer the polymerase and exonuclease activities of the Klenow LargeFragment Polymerase can be used to fill in 5′ overhangs and remove 3′overhangs on the nucleic acid molecules. In some embodiments, aphosphate is added on the 5′ end of the repaired nucleic acid moleculesusing Polynucleotide Kinase (PNK) and reaction conditions a skilledartisan will understand. In further illustrative embodiments, a singlenucleotide or multiple nucleotides is added to one strand of a doublestranded molecule to generate a “sticky end.” For example, an adenosine(A) can be appended on the 3′ ends of the nucleic acid molecules(A-tailing). In some embodiments, other sticky ends are used other thanan A overhang. In some embodiments, other adapters are added, forexample looped ligation adapters. In some embodiments, adapters areadded during a PCR step. In any of the embodiments of the presentteachings, none, all, or any combination of these modifications arecarried out. Many kits and methods are known in the art for generatingpopulations of templates nucleic acid molecules for subsequentsequencing. Such kits would typically be modified to include adaptersthat are customized for the amplification and sequencing steps of themethods and compositions of the present teachings. Adapter ligation canalso be performed using commercially available kits such as the ligationkit found in the Agilent SureSelect kit (Agilent).

In some embodiments, the amplification methods optionally include atarget enrichment step before, during, or after the library preparationand before a pre-seeding reaction. Target nucleic acid molecules,including target loci or regions of interest, can be enriched, forexample, through multiplex nucleic acid amplification or hybridization.A variety of methods are known in the art to perform multiplex nucleicacid amplification to generate amplicons, such as multiplex PCR, and canbe used in any of the embodiments of the present teachings. Enrichmentby any method can be followed by a universal amplification reactionbefore the template nucleic acid molecules are added to a pre-seedingreaction mixture. Any of the embodiments of the present teachingsinclude enriching a plurality of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000,2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 targetnucleic acid molecules, target loci, or regions of interest. In any ofthe disclosed embodiments, the target loci or regions of interest are atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700, 800,900, or 1,000 nucleotides in length and include a portion of or theentirety of the template nucleic acid molecule. In other embodiments,the target loci or regions of interest are between about 1 and 10,000nucleotides in length, for example between about 2 and 5,000nucleotides, between about 2 and 3,000 nucleotides, or between about 2and 2,000 nucleotides in length. In any of the embodiments of thepresent teachings, the multiplex nucleic acid amplification includesgenerating at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200,250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 copies of each targetnucleic acid molecule, target locus, or region of interest. In any ofthe disclosed embodiments, methods, as well as related compositions,systems, kits, and apparatuses, for pre-seeding, include generating apopulation of pre-seeded solid supports using the amplicons from themultiplex nucleic acid amplification in a pre-seeding reaction.

In some embodiments, after the library preparation and optionalenrichment step, the library of template nucleic acid molecules istemplated onto one or more supports. In the present disclosure, the oneor more supports are typically templated in two reactions, a pre-seedingreaction to generate pre-seeded solid supports and a templating reactionusing the one or more pre-seeded supports to further amplify theattached template nucleic acid molecules. The pre-seeding reaction istypically an amplification reaction and can be performed using a varietyof methods a skilled artisan will understand. For example, thepre-seeding reaction can be performed in an RPA reaction, a templatewalking reaction, or a PCR. In an RPA reaction, template nucleic acidmolecules are amplified using a recombinase, polymerase, and optionallya recombinase accessory protein in the presence of primers andnucleotides. The recombinase and optionally the recombinase accessoryprotein can dissociate at least a portion of a double-stranded templatenucleic acid molecules to allow primers to hybridize that the polymerasecan then bind to initiate replication. In some embodiments, therecombinase accessory protein is a single-stranded binding protein (SSB)that prevents the re-hybridization of dissociated template nucleic acidmolecules. Typically, RPA reactions are performed at isothermaltemperatures. In a template walking reaction, template nucleic acidmolecules are amplified using a polymerase in the presence of primersand nucleotides in reaction conditions that allow at least a portion ofdouble-stranded template nucleic acid molecules to dissociate such thatprimers can hybridize and the polymerase can then bind to initiatereplication. In PCR, the double-stranded template nucleic acid moleculesare dissociated by thermal cycling. After cooling, primers bind tocomplementary sequences and can be used for replication by thepolymerase. In any of the aspects of the present teachings, thepre-seeding reaction is performed in a pre-seeding reaction mixture,which is formed with the components necessary for amplification of thetemplate nucleic acid molecules. In any of the disclosed aspects, thepre-seeding reaction mixture includes some or all of the following: apopulation of template nucleic acid molecules, a polymerase, one or moresolid supports with a population of attached first primers, nucleotides,and a cofactor such as a divalent cation. In some embodiments, thepre-seeding reaction mixture further includes a second primer andoptionally a diffusion-limiting agent. In some embodiments, thepopulation of template nucleic acid molecules comprise template nucleicacid molecules joined to at least one adaptor sequence which hybridizeto the first or second primers. In some embodiments, the reactionmixture forms an emulsion, as in emulsion RPA or emulsion PCR. Inpre-seeding reactions carried out by RPA reactions, the pre-seedingreaction mixture includes a recombinase and optionally a recombinaseaccessory protein. The various components of the reaction mixture arediscussed in further detail herein.

In some embodiments, the pre-seeding reaction mixtures include apopulation of template nucleic acid molecules that is typically derivedfrom the library preparation or target enrichment. In some embodiments,template nucleic acid molecules or populations of template nucleic acidmolecules are at least some, and typically all members of a library oftemplate nucleic acid molecules. In some embodiments, the pre-seedingreaction mixture includes at least one template nucleic acid molecule.In some embodiments, the pre-seeding reaction mixture includes at leasttwo template nucleic acid molecules with different sequences. Inillustrative embodiments, the pre-seeding reaction mixture includes apopulation of template nucleic acid molecules with different sequences.In some embodiments, the pre-seeding reaction mixture includes apopulation of substantially monoclonal template nucleic acid molecules.In any of the embodiments of the present teachings, the template nucleicacid molecules are polynucleotides as they are alternatively referred toherein (the template nucleic acid molecules are interchangeably referredto herein as a template or a nucleic acid template or a polynucleotidetemplate). In various embodiments, the template nucleic acid moleculesare polymers of deoxyribonucleotides, ribonucleotides, and/or analogsthereof. In some embodiments, the polynucleotides arenaturally-occurring, synthetic, recombinant, cloned, amplified,unamplified, or archived (e.g., preserved) forms. In some embodiments,the polynucleotides are DNA, cDNA, RNA, or chimeric RNA/DNA, and nucleicacid analogs.

The template nucleic acid molecule to be amplified can bedouble-stranded, or is rendered at least partially double-stranded usingappropriate procedures prior to the pre-seeding reaction. In someembodiments, the template is linear. Alternatively, the template can becircular or include a combination of linear and circular regions. Insome embodiments, the amplifying includes forming a partially denaturedtemplate. For example, the amplification can include partiallydenaturing a double-stranded template nucleic acid molecule. Optionally,partially denaturing includes subjecting the double-stranded templatenucleic acid molecule to partially denaturing conditions. In someembodiments, the partially denatured template includes a single-strandedportion and a double-stranded portion. In some embodiments, thesingle-stranded portion includes the first primer binding sequence. Insome embodiments, the single-stranded portion includes the second primerbinding sequence. In some embodiments, the single-stranded portionincludes both the first primer binding sequence and the second primerbinding sequence.

Optionally, the double-stranded template nucleic acid molecule includesa forward strand. The double-stranded template nucleic acid molecule canfurther include a reverse strand. The forward strand optionally includesa first primer binding sequence. The reverse strand optionally includesa second primer binding sequence. The primer binding sequences aretypically attached during the library preparation as disclosed above. Insome embodiments, the template nucleic acid molecule already includes afirst primer binding sequence and optionally a second primer bindingsequence. Alternatively, the template nucleic acid molecule optionallydoes not originally include a primer binding sequence, and the librarypreparation can optionally include attaching or introducing a primerbinding sequence to the template as disclosed above.

In some embodiments, the template nucleic acid molecules includesingle-stranded or double-stranded polynucleotides, or a mixture ofboth. In some embodiments, the template nucleic acid molecules includepolynucleotides with the same or different nucleotide sequences. In someembodiments, the template nucleic acid molecules include polynucleotideshaving the same or different lengths. In various embodiments, thepre-seeding reaction mixture includes between about 2 and 10¹² differenttemplate nucleic acid molecules, for example between about 2 and 10¹¹different template nucleic acid molecules, between about 2 and 10¹⁰different template nucleic acid molecules, between about 2 and 10⁹different template nucleic acid molecules, between about 2 and 10⁸different template nucleic acid molecules, between about 2 and 10⁷different template nucleic acid molecules, between about 2 and 10⁶different template nucleic acid molecules, or between about 2 and500,000 different template nucleic acid molecules. In any of thedisclosed embodiments, the reaction mixture includes between 5×10⁶ and10¹⁰ solid supports. The solid supports can have a smallestcross-sectional length (e.g., diameter) of 50 microns or less,preferably 10 microns or less, 3 microns or less, approximately 1 micronor less, approximately 0.5 microns or less, e.g., approximately 0.1,0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer, about1-10 nanometer, about 10-100 nanometers, or about 100-500 nanometers).In any of the embodiments of the present teachings, the volumes of thepre-seeding reaction mixtures and/or templating reaction mixtures arebetween about 50 and 2,000 μl, for example between about 50 and 1,500μl, between about 50 and 1,000 μl, between about 50 and 500 μl, betweenabout 50 and 250 μl, or between about 50 and 150 μl.

The pre-seeding reaction is performed to generate one or more pre-seededsupports. Accordingly, in any of the disclosed aspects, the pre-seedingreaction mixture can include one or more solid or semi-solid supports towhich the template nucleic acid molecules are attached. As used herein,solid or semi-solid supports can also refer to one support with sites ofattachment that can be distinctly analyzed in downstream sequencingmethods. In illustrative embodiments, the one or more supports include apopulation of attached substantially identical first primers. In someembodiments, at least one template nucleic acid molecule in the reactionmixture includes a first primer binding sequence. The first primerbinding sequence can be substantially identical or substantiallycomplementary to the sequence of the first primer. In some embodiments,at least one, some, or all of the supports include a population of firstprimers that are substantially identical to each other. In someembodiments, all of the primers on the supports are substantiallyidentical to each other or all include a substantially identical firstprimer sequence. In some embodiments, at least one of the supportsincludes two or more different primers attached thereto. For example,the at least one support can include a population of a first primer anda population of a second primer. The support can be attached to auniversal primer. The universal primer optionally hybridizes (or iscapable of hybridizing) to all, or substantially all, of the templatenucleic acid molecules within the reaction mixture. The reaction mixturecan include a first support covalently attached to a firsttarget-specific primer and a second support covalently attached to asecond target-specific primer, wherein the first and secondtarget-specific primers are different from each other. Optionally, thefirst target-specific primer is substantially complementary to a firsttarget nucleic acid sequence and the second target-specific primer issubstantially complementary to a second target nucleic acid sequence,and wherein the first and second target nucleic acid sequences aredifferent.

In some embodiments, two or more different template nucleic acidmolecules having a first primer binding sequence are included in thepre-seeding reaction mixture. In some embodiments, the at least twodifferent template nucleic acid molecules are amplified directly onto asupport such as a site on a support that includes a plurality of sites,a bead or microparticle, or a reaction chamber of an array. The templatenucleic acid molecules can be pre-seeded in bulk in solution and thendistributed into an array of wells or reaction sites on a solid support.Alternatively, solid supports can be distributed into an array of wellsand template nucleic acid molecules can be pre-seeded on the solidsupports while they are held in place in an array of wells. In someembodiments, methods for nucleic acid amplification include one or moresurfaces.

In some embodiments, a surface has attached a population of firstprimers, the first primers of the population sharing a common firstprimer sequence. In some embodiments, a surface has attached apopulation of first primers and a population of second primers, thefirst primers of the population sharing a common first primer sequenceand the second primers of the population of second primers sharing acommon second primer sequence. In some embodiments, the surface hasimmobilized thereon a population of first primers. In other embodiments,the surface has immobilized thereon a population of first primers and apopulation of second primers.

A support or surface can be coated with an acrylamide, carboxylic, oramine compound for attaching a nucleic acid molecule (e.g., a firstprimer or second primer). In some embodiments, an amino-modified nucleicacid molecule (e.g., primer) is attached to a support that is coatedwith a carboxylic acid. In some embodiments, an amino-modified nucleicacid molecule is reacted with EDC (or EDAC) for attachment to acarboxylic acid coated surface (with or without NHS). A first primer canbe attached to an acrylamide compound coating on a surface. Particlescan be coated with an avidin-like compound (e.g., streptavidin) forbinding biotinylated nucleic acids.

In some embodiments, the reaction mixture includes multiple differentsurfaces, for example, the pre-seeding reaction mixture includes one ormore beads (such as particles, nanoparticles, microparticles, and thelike) and at least two different template nucleic acid molecules areamplified onto different beads, thereby forming at least two differentbeads, each of which is attached to a different template nucleic acidmolecule. In some embodiments, the pre-seeding reaction mixture includesa single surface (for example, a planar-like surface, a flowcell, orarray of reaction chambers) and at least two different template nucleicacid molecules are amplified onto two different regions or locations onthe surface, thereby forming a single surface attached to two or moretemplate nucleic acid molecules.

In some embodiments, a surface of a solid support is porous, semi-porousor non-porous. In some embodiments, a surface is a planar surface, aswell as concave, convex, or any combination thereof. In someembodiments, a surface is a bead, particle, microparticle, sphere,filter, flowcell, well, groove, channel reservoir, gel, or inner wall ofa capillary. In some embodiments, a surface includes texture (e.g.,etched, cavitated, pores, three-dimensional scaffolds or bumps).

In some embodiments, a surface of a solid support is a magnetic orparamagnetic bead (e.g., magnetic or paramagnetic nanoparticles ormicroparticles). In some embodiments, paramagnetic microparticles areparamagnetic beads attached with streptavidin (e.g., Dynabeads™ M-270from Invitrogen, Carlsbad, Calif.). Particles can have an iron core, orcan be a hydrogel or agarose (e.g., Sepharose™).

In some embodiments, the surface includes the surface of a bead. In someembodiments, a bead is a polymer material. For example, a bead can be agel, hydrogel, or acrylamide polymers. A bead can be porous. Particlescan have cavitation or pores, or can include three-dimensionalscaffolds. In some embodiments, particles are Ion Sphere™ Particles(ThermoFisher Scientific, Waltham, Mass.).

In general, the polymeric particle or bead support can be treated toinclude a biomolecule, including nucleosides, nucleotides, nucleic acids(oligonucleotides and polynucleotides), polypeptides, saccharides,polysaccharides, lipids, or derivatives or analogs thereof. For example,a polymeric particle can bind or attach to a biomolecule. A terminal endor any internal portion of a biomolecule can bind or attach to apolymeric particle. A polymeric particle can bind or attach to abiomolecule using linking chemistries. A linking chemistry includescovalent or non-covalent bonds, including an ionic bond, hydrogen bond,affinity bond, dipole-dipole bond, van der Waals bond, and hydrophobicbond. A linking chemistry includes affinity between binding partners,for example between: an avidin moiety and a biotin moiety; an antigenicepitope and an antibody or immunologically reactive fragment thereof; anantibody and a hapten; a digoxigen moiety and an anti-digoxigenantibody; a fluorescein moiety and an anti-fluorescein antibody; anoperator and a repressor; a nuclease and a nucleotide; a lectin and apolysaccharide; a steroid and a steroid-binding protein; an activecompound and an active compound receptor; a hormone and a hormonereceptor; an enzyme and a substrate; an immunoglobulin and protein A; oran oligonucleotide or polynucleotide and its corresponding complement.

In particular, a solid phase support, such a bead support, can includecopies of polynucleotides. In a particular example, polymeric particlescan be used as a support for polynucleotides during sequencingtechniques. For example, such hydrophilic particles can immobilize apolynucleotide for sequencing using fluorescent sequencing techniques.In another example, the hydrophilic particles can immobilize a pluralityof copies of a polynucleotide for sequencing using ion-sensingtechniques. Alternatively, the above described treatments can improvepolymer matrix bonding to a surface of a sensor array. The polymermatrices can capture analytes, such as polynucleotides for sequencing.

In some embodiments, one or more nucleic acid templates are immobilizedonto one or more supports. Template nucleic acid molecules may beimmobilized on the support by any method including but not limited tophysical adsorption, by ionic or covalent bond formation, orcombinations thereof. A solid support may include a polymeric, a glass,or a metallic material. Examples of solid supports include a membrane, aplanar surface, a microtiter plate, a bead, a filter, a test strip, aslide, a cover slip, and a test tube. A solid support means any solidphase material upon which an oligomer is synthesized, attached, ligated,or otherwise immobilized. A support can optionally include a “resin”,“phase”, “surface”, and “support”. A support may be composed of organicpolymers such as polystyrene, polyethylene, polypropylene,polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well asco-polymers and grafts thereof. A support may also be inorganic, such asglass, silica, controlled-pore-glass (CPG), or reverse-phase silica. Theconfiguration of a support may be in the form of beads, spheres,particles, granules, a gel, or a surface. Surfaces may be planar,substantially planar, or non-planar. Supports may be porous ornon-porous, and may have swelling or non-swelling characteristics. Asupport can be shaped to include one or more wells, depressions or othercontainers, vessels, features, or locations. One or more supports may beconfigured in an array at various locations. A support is optionallyaddressable (e.g., for robotic delivery of reagents), or by detectionmeans including scanning by laser illumination and confocal ordeflective light gathering. A support (e.g., a bead) can be placedwithin or on another support (e.g., within a well of a second support).In some embodiments, a support is an Ion Sphere Particle.

In some embodiments, the solid support is a “microparticle,” “bead,”“microbead,” etc., (optionally but not necessarily spherical in shape)having a smallest cross-sectional length (e.g., diameter) of 50 micronsor less, preferably 10 microns or less, 3 microns or less, approximately1 micron or less, approximately 0.5 microns or less, e.g., approximately0.1, 0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer,about 1-10 nanometer, about 10-100 nanometers, or about 100-500nanometers). Microparticles (e.g., Dynabeads from Dynal, Oslo, Norway)may be made of a variety of inorganic or organic materials including,but not limited to, glass (e.g., controlled pore glass), silica,zirconia, cross-linked polystyrene, polyacrylate,polymethylmethacrylate, titanium dioxide, latex, polystyrene, etc.Magnetization can facilitate collection and concentration of themicroparticle-attached reagents (e.g., polynucleotides or ligases) afteramplification, and can also facilitate additional steps (e.g., washes,reagent removal, etc.). In certain embodiments, a population ofmicroparticles having different shapes sizes and/or colors is used. Themicroparticles can optionally be encoded, e.g., with quantum dots suchthat each microparticle or group of microparticles can be individuallyor uniquely identified.

In some embodiments, a bead surface is functionalized for attaching apopulation of first primers. In some embodiments, a bead is any sizethat can fit into a reaction chamber. For example, one bead can fit in areaction chamber. In some embodiments, more than one bead fit in areaction chamber. In some embodiments, the smallest cross-sectionallength of a bead (e.g., diameter) is about 50 microns or less, or about10 microns or less, or about 3 microns or less, approximately 1 micronor less, approximately 0.5 microns or less, e.g., approximately 0.1,0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer, about1-10 nanometer, about 10-100 nanometers, or about 100-500 nanometers).

In some embodiments, the two or more different template nucleic acidmolecules are localized, deposited, or positioned at different sitesprior to the pre-seeding reaction. In some embodiments, the two or moredifferent template nucleic acid molecules are pre-seeded in solution,optionally within a single pre-seeding reaction mixture, and theresulting two or more substantially monoclonal populations of templatenucleic acid molecules are then localized, deposited, or positioned atdifferent sites following such amplification. The different sites areoptionally members of an array of sites. The array can include atwo-dimensional array of sites on a surface (e.g., of a flowcell,electronic device, transistor chip, reaction chamber, channel, and thelike), or a three-dimensional array of sites within a matrix or othermedium (e.g., solid, semi-solid, liquid, fluid, and the like).

In some embodiments, the methods for pre-seeding template nucleic acidmolecules onto one or more supports, as well as the templatingreactions, typically use one or more enzymes capable of polymerization.In any of the embodiments of the present teachings, the one or moreenzymes capable of polymerization include at least one polymerase. Insome embodiments, the at least one polymerase includes a thermostable orthermolabile polymerase. In some embodiments, the at least onepolymerase includes a biologically active fragment of a DNA or RNApolymerase that maintains sufficient catalytic activity to polymerize orincorporate at least one nucleotide under any suitable conditions. Invarious embodiments, the at least one polymerase includes a mutated DNAor RNA polymerase that maintains sufficient catalytic activity toperform nucleotide polymerization under any suitable conditions. Invarious embodiments, the at least one polymerase includes one or moreamino acid mutations that maintains sufficient catalytic activity toperform polymerization. The polymerase optionally can have, or lack,exonuclease activity. In some embodiments, the polymerase has 5′ to 3′exonuclease activity, 3′ to 5′ exonuclease activity, or both.Optionally, the polymerase lacks any one or more of such exonucleaseactivities. In some embodiments, the polymerase has strand-displacingactivity. Examples of useful strand-displacing polymerases includeBacteriophage Φ29 DNA polymerase and Bst DNA polymerase.

In some embodiments, a polymerase includes any enzyme or fragment orsubunit thereof, that can catalyze polymerization of nucleotides and/ornucleotide analogs. In some embodiments, a polymerase requires anextendible 3′ end. For example, a polymerase requires a terminal 3′ OHof a nucleic acid primer to initiate nucleotide polymerization. Thepolymerase can be other than a thermostable polymerase. For example, thepolymerase can be active at 37° C. and/or more active at 37° C. than at50° C., 60° C., 70° C. or higher. In various embodiments, the polymerasecan be active and/or more active at 42° C., 45° C., 50° C., 55° C., or60° C. than at 37° C.

A polymerase can include any enzyme that can catalyze the polymerizationof nucleotides (including analogs thereof) into a nucleic acid strand.Typically, but not necessarily, such nucleotide polymerization can occurin a template-dependent fashion. In some embodiments, a polymerase is ahigh-fidelity polymerase. Such polymerases can include, withoutlimitation, naturally-occurring polymerases and any subunits andtruncations thereof, mutant polymerases, variant polymerases,recombinant, fusion or otherwise engineered polymerases,chemically-modified polymerases, synthetic molecules or assemblies, andany analogs, derivatives, or fragments thereof that retain the abilityto catalyze such polymerization. Optionally, the polymerase is a mutantpolymerase with one or more mutations involving the replacement of oneor more amino acids with other amino acids, the insertion or deletion ofone or more amino acids from the polymerase, or the linkage of parts oftwo or more polymerases. The term “polymerase” and its variants, as usedherein, also refers to fusion proteins including at least two portionslinked to each other, where the first portion can include a peptide thatcan catalyze the polymerization of nucleotides into a nucleic acidstrand and is linked to a second portion that can include a secondpolypeptide, such as, for example, a reporter enzyme or aprocessivity-enhancing domain. Typically, the polymerase includes one ormore active sites at which nucleotide binding and/or catalysis ofnucleotide polymerization can occur. In some embodiments, a polymerasecan include or lack other enzymatic activities, such as for example, 3′to 5′ exonuclease activity or 5′ to 3′ exonuclease activity. In someembodiments, a polymerase is isolated from a cell, or generated usingrecombinant DNA technology or chemical synthesis methods. In any of theembodiments of the present teachings, a polymerase is expressed inprokaryote, eukaryote, viral, or phage organisms. In variousembodiments, a polymerase is a DNA polymerase and include withoutlimitation bacterial DNA polymerases, eukaryotic DNA polymerases,archaeal DNA polymerases, viral DNA polymerases, and phage DNApolymerases. In various embodiments, the expressed polymerase ispurified using methods known in the art. In some embodiments, apolymerase is post-translationally modified proteins or fragmentsthereof.

In some embodiments, the polymerase includes any one or morepolymerases, or biologically active fragments of a polymerase, asdescribed in U.S. Patent Publ. No. 2011/0262903 to Davidson et al.,published Oct. 27, 2011, and/or International PCT Publ. No. WO2013/023176 to Vander Horn et al., published Feb. 14, 2013, hereinincorporated by reference in their entireties.

In some embodiments, a polymerase is a replicase, DNA-dependentpolymerase, primases, RNA-dependent polymerase (including RNA-dependentDNA polymerases such as, for example, reverse transcriptases), athermo-labile polymerase, or a thermo-stable polymerase. In someembodiments, a polymerase is any Family A or B type polymerase. Manytypes of Family A (e.g., E. coli Pol I), B (e.g., E. coli Pol II), C(e.g., E. coli Pol III), D (e.g., Euryarchaeotic Pol II), X (e.g., humanPol beta), and Y (e.g., E. coli UmuC/DinB and eukaryotic RAD30/xerodermapigmentosum variants) polymerases are described in Rothwell and Watsman2005 Advances in Protein Chemistry 71:401-440. In some embodiments, apolymerase is a T3, T5, T7, or SP6 RNA polymerase.

In some embodiments, nucleic acid amplification reactions are conductedwith one type or a mixture of polymerases and/or ligases. In someembodiments, nucleic acid amplification reactions are conducted with alow-fidelity or high-fidelity polymerase or without regard to fidelity.

An exemplary polymerase is Bst DNA Polymerase (Exonuclease Minus), is a67 kDa Bacillus stearothermophilus DNA Polymerase protein (largefragment), exemplified in accession number 2BDP_A, which has 5′ to 3′polymerase activity and strand displacement activity but lacks 3′ to 5′exonuclease activity. Other polymerases include Taq DNA polymerase Ifrom Thermus aquaticus (exemplified by accession number 1TAQ), Eco DNApolymerase I from Escherichia coli (accession number P00582), Aea DNApolymerase I from Aquifex aeolicus (accession number 067779), orfunctional fragments or variants thereof, e.g., with at least 80%, 85%,90%, 95% or 99% sequence identity at the nucleotide level.

In illustrative embodiments, the DNA polymerase is a Bsu DNA polymerase(large fragment (NEB)). Bsu DNA Polymerase I, Large Fragment retains the5′ to 3′ polymerase activity of the Bacillus subtilis DNA polymerase I(1), but lacks the 5′ to 3′ exonuclease domain. In certain embodiments,the Bsu DNA Polymerase large fragment lacks 3′ to 5′ exonucleaseactivity. In various embodiments, Bsu DNA Polymerase large fragment hasoptimal activity at 37° C.

In certain illustrative embodiments, especially where the pre-seedingreaction is an RPA reaction, the one or more enzymes capable ofpolymerization include a T5 or T7 DNA polymerase. In some embodiments,the one or more enzymes capable of polymerization include a T5 or T7 DNApolymerase having one or more amino acid mutations that reduce the 3′ to5′ exonuclease activity. In some embodiments, the T5 or T7 DNApolymerase having one or more amino acid mutations that reduce the 3 to5′ exonuclease activity does not contain an amino acid mutation thatdisrupts the processivity of the T5 or T7 DNA polymerase. In someembodiments, the T5 or T7 DNA polymerase includes one or more amino acidmutations that eliminate detectable 3′ to 5′ exonuclease activity; andwherein the one or more amino acid mutations do not disrupt processivityof the T5 or T7 DNA polymerase. In certain illustrative embodiments, thepre-seeding reaction mixture includes a Sau polymerase, T7 DNApolymerase with reduced 3′ to 5′ exonuclease activity, Bsu polymerase,or a combination thereof. These polymerases that are especially wellsuited for an RPA reaction, are well-suited not only for the pre-seedingreaction, but the templating reaction as well.

In some embodiments, the one or more enzymes capable of polymerizationinclude any suitable RNA polymerase. Suitable RNA polymerases include,without limitation, T3, T5, T7, and SP6 RNA polymerases.

In various embodiments, template nucleic acid molecules used in any ofthe methods of the present teachings, including the pre-seeding reactionand the templating reaction, typically include a first primer bindingsequence (“forward”) and optionally a second primer binding sequence(“reverse”). Accordingly, the pre-seeding reaction mixture and thetemplating reaction include a population of first primers and optionallya population of second primers that bind the forward primer binding andreverse primer binding sequences, respectively. In some embodiments, thefirst and second primers are referred to as a primer pair. In someembodiments, the first primers and/or the second primers are typicallyuniversal primers. The first primer can bind to either the forwardprimer binding sequence or the reverse primer binding sequence and thesecond primer can bind to either the forward primer binding sequence orthe reverse primer binding sequence.

In any of the disclosed embodiments, the reaction mixture includes apopulation of first primers and a population of second primers that bindto sequences within the template nucleic acid molecules. The populationof first primers can be identical copies or different sequences. Thepopulation of second primers can be identical copies or differentsequences. However, the population of first primers and optional secondprimers, are typically a universal primer and all copies are identical.Thus, in illustrative embodiments, the population of first primers andthe population of second primers are both be universal primers that binduniversal primer binding sequences on the template nucleic acidmolecules. In other embodiments, both the population of first primersand the population of second primers are target-specific primers. Thepopulation of first primers and the population of second primers canhave the same or different sequences. In any of embodiments of thepresent teachings, the population of first primers and/or the populationof second primers are attached to one or more supports prior toincubation with the pre-seeding reaction mixture. In other embodiments,the population of first primers and/or the population of second primersare in solution during incubation with the pre-seeding reaction mixture.In illustrative embodiments, the population of first primers is attachedto one or more supports prior to incubation with the pre-seedingreaction mixture and the population of second primers is typically insolution during incubation with the pre-seeding reaction mixture.

Thus, in these illustrative embodiments that include a population ofimmobilized first primers and a population of second primers insolution, not to be limited by theory, during the pre-seeding reaction,a template nucleic acid is at least partially denatured, and the firstprimer binding site on the template binds to a first template attachedto a solid support. The first primer is used by a polymerase to generatea complementary strand to one strand of the template nucleic acid. Thatcomplementary strand is now covalently attached to the solid supportthrough the primer. A second primer in solution is in a complex with therecombinase and binds to a primer binding site on the complementarystrand, thus partially denaturing the bound template nucleic acidmolecule. A polymerase use the primer to synthesize a new strand,identical to the original template nucleic acid strand. This strand isthen believed to be partially denatured by the binding of the complex ofa recombinase and a nearby first primer attached to the solid support,and the polymerase synthesizes another complementary strand. Throughrepeated steps of this process, a substantially monoclonal population oftemplate nucleic acid molecules is generated during the pre-seedingreaction, and further amplified during a templating reaction.

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses, in which theprimers typically have a free 3′ hydroxyl. In some embodiments, theprimers are polymers of ribonucleotides, deoxyribonucleotides, oranalogs thereof. In some embodiments, primers are naturally-occurring,synthetic, recombinant, cloned, amplified, or unamplified forms. In someembodiments, the primers include phosphodiester linkages between allnucleotides. In any of the embodiments of the present teachings, theprimers are between about 5 and 100 nucleotides in length, for examplebetween about 5 and 80 nucleotides in length, between about 5 and 60nucleotides in length, between about 5 and 40 nucleotides in length,between about 10 and 75 nucleotides in length, between about 15 and 75nucleotides in length, or between about 20 and 50 nucleotides in length.

In some embodiments, at least one of the primers has modifications. Forexample, ribonucleotide, deoxyribonucleotides, or analogs thereof canhave biotin or azide attached to them. In some embodiments, theribonucleotide, deoxyribonucleotides, or analogs thereof have attachedfluorophores, phosphorylation, or spacers. In some embodiments, theprimers are blocked and/or are fusion primers or fusion polynucleotideswhere different regions of the primers or polynucleotides are designedto bind to one of two primer binding sites and/or are designed to bebound by one of two primers.

In some embodiments, the primers are blocked primers to preventextension from the 3′ end of the primer. In some embodiments, theblocked primers are tailed primers wherein the 5′ end includes asequence that is non-complementary to the template nucleic acidmolecules. This 5′ sequence can be used as a template for primerextension reactions. In some embodiments, the primers are blockedwherein the 5′ domain is 15 to 30 nucleotides in length. In someembodiments, the primers include a blocking moiety at their 5′ or 3′end, or at the 5′ and 3′ ends. In a reaction that involves primerextension (e.g., pre-seeding amplification), the blocking moiety at the3′ end of the blocked fusion primers can reduce the level ofprimer-dimer formation. In certain embodiments, the pre-seeding reactionmixture includes a blocked primer wherein the 3′ domain is 14 to 25nucleotides in length. In other embodiments, the 3′ domain is 15 to 25nucleotides in length. In still other embodiments, the 5′ domain is atleast 15 nucleotides and the 3′ domain is at least 10 nucleotides,wherein the length of the primer does not exceed 100, 90, 80, 75, 70,60, or 50 nucleotides. In embodiments, a 3′ nucleotide of the 3′ domainof the forward primer is mismatched to the forward primer bindingsequence. In embodiments, the ribobase separating the 5′ domain and the3′ domain of the blocked primer includes rU, rG, rC, or rA. In certainembodiments, the ribobase separating the 5′ domain and the 3′ domain ofthe blocked primer includes rC. In any of the embodiments of the presentteachings, the 3′ domain of the blocked primers is 14 to 20 nucleotidesin length and the ribobase is rU, rG, rC or rA.

In any of the embodiments of the present teachings, the reaction mixtureincludes an enzyme to remove a portion of the blocked primers to leave afree 3′ OH. After removing the blocked end of the primer, a polymerasecan initiate replication from the free 3′ OH to begin replicating thetemplate strand. In some embodiments, this enzyme is an RNase,especially RNase H. A skilled artisan will recognize other compositionsof blocked primers to use and suitable enzymes for removing a portion ofthe blocked primers.

The pre-seeding reaction mixture, as well an any other amplificationreaction in the methods provided here, including the templating reactionmixture, typically includes a source of nucleotides, or analogs thereof,that is used by the polymerase as substrates for an extension reaction.In any of the embodiments of the present teachings, the pre-seedingreaction mixture typically includes nucleotides (dNTPs) for strandextension of the template nucleic acid molecules resulting in asubstantially monoclonal population of the template nucleic acidmolecule sequence attached to one or more supports. In some embodiments,nucleotides are not extrinsically labeled. For example, the nucleotidescan be naturally-occurring nucleotides or synthetic analogs that do notinclude fluorescent moieties, dyes, or other extrinsic opticallydetectable labels. Optionally, the nucleotides do not include groupsthat terminate nucleic acid synthesis (e.g., dideoxy groups, reversibleterminators, and the like). In other embodiments, the nucleotidesinclude a label or tag.

In some embodiments, methods for nucleic acid amplification includes atleast one cofactor, for example a cofactor that enhances activity of aDNA or RNA polymerase. In some embodiments, a cofactor includes one ormore divalent cations. Examples of divalent cations include magnesium,manganese, and calcium. In various embodiments, the pre-seeding reactionmixture includes a buffer containing one or more divalent cation. Inillustrative embodiments, the buffer contains magnesium or manganeseions. In any of the embodiments of the present teachings, thepre-seeding reaction or the templating reaction is initiated by theaddition of a cofactor, especially a divalent cation. In someembodiments, the pre-seeding reaction mixture used herein for nucleicacid amplification may include at least one cofactor for recombinaseassembly on nucleic acids or for homologous nucleic acid pairing. Insome embodiments, a cofactor includes any form of ATP including ATP andATPγS. In some embodiments, methods for nucleic acid amplificationincludes at least one cofactor that regenerates ATP. For example, acofactor can include an enzyme system that converts ADP to ATP. In someembodiments, a cofactor enzyme system is phosphocreatine and creatinekinase.

In any aspects of the present teachings, the pre-seeding reactionmixture includes components to partially denature template nucleic acidmolecules. In some embodiments, partially denaturing conditions includetreating or contacting the template nucleic acid molecules to beamplified with one or more enzymes that are capable of partiallydenaturing the nucleic acid template, optionally in a sequence-specificor sequence-directed manner, as in an RPA reaction. In some embodiments,at least one enzyme catalyzes strand invasion and/or unwinding,optionally in a sequence-specific manner. Optionally, the one or moreenzymes include one or more enzymes selected from the following:recombinases, topoisomerases, and helicases. In some embodiments,partially denaturing the template includes contacting the template witha recombinase and forming a nucleoprotein complex including therecombinase. Optionally, the template nucleic acid molecule is contactedwith a recombinase in the presence of a first and optionally a secondprimer. Partially denaturing can include catalyzing strand exchangeusing the recombinase and hybridizing the first primer to the firstprimer binding sequence (or hybridizing the second primer to the secondprimer binding sequence). In some embodiments, partially denaturingincludes performing strand exchange and hybridizing both the firstprimer to the first primer binding sequence and the second primer to thesecond primer binding sequence using the recombinase.

In some embodiments, partially denaturing the template nucleic acidmolecule includes contacting the template with one or more recombinasesor nucleoprotein complexes. At least one of the nucleoprotein complexescan include a recombinase. At least one of the nucleoprotein complexescan include a primer (e.g., a first primer or a second primer, or aprimer including a sequence complementary to a corresponding primerbinding sequence in the template). In some embodiments, partiallydenaturing the template includes contacting the template with anucleoprotein complex including a primer. Partially denaturing caninclude hybridizing the primer of the nucleoprotein complex to thecorresponding primer binding sequence in the template, thereby forming aprimer-template duplex. In some embodiments, partially denaturing thetemplate nucleic acid molecule includes contacting the template with afirst nucleoprotein complex including a first primer. Partiallydenaturing can include hybridizing the first primer of the firstnucleoprotein complex to the first primer binding sequence of theforward strand, thereby forming a first primer-template duplex. In someembodiments, partially denaturing the template includes contacting thetemplate with a second nucleoprotein complex including a second primer.Partially denaturing can include hybridizing the second primer of thesecond nucleoprotein complex to the second primer binding sequence ofthe reverse strand, thereby forming a second primer-template duplex.

Accordingly, the pre-seeding reaction mixtures of the presentdisclosure, and a templating reaction of the present disclosure, includea recombinase and partial denaturation and/or amplification, includingany one or more steps or methods described herein, can be achieved usinga recombinase and optionally a recombinase accessory protein. Therecombinase can include any agent that is capable of inducing, orincreasing the frequency of occurrence, of a recombination event. Arecombination event includes any event whereby two differentpolynucleotides strands are recombined with each other. Recombinationcan include homologous recombination. The recombinase optionally canassociate with (e.g., bind) a first primer. In some embodiments, anenzyme that catalyzes homologous recombination can form a nucleoproteincomplex by binding a single-stranded template nucleic acid molecule. Insome embodiments, a homologous recombination enzyme, as part of anucleoprotein complex, can bind a homologous portion of adouble-stranded polynucleotide. In some embodiments, the homologousportion of the polynucleotide hybridizes to at least a portion of thefirst primer. In some embodiments, the homologous portion of thepolynucleotide is partially or completely complementary to at least aportion of the first primer. Suitable recombinases include RecA and itsprokaryotic or eukaryotic homologues, or functional fragments orvariants thereof, optionally in combination with one or moresingle-strand binding proteins (SSBs). In certain embodiments, therecombinase optionally coats ssDNA to form a nucleoprotein filamentstrand which invades a double-stranded region of homology on a template.

In some embodiments, a homologous recombination enzyme catalyzes strandinvasion by forming a nucleoprotein complex and binding to a homologousportion of a double-stranded polynucleotide to form a recombinationintermediate having a triple-strand structure (D-loop formation) (U.S.Pat. No. 5,223,414 to Zarling, U.S. Pat. Nos. 5,273,881 and 5,670,316both to Sena, and U.S. Pat. Nos. 7,270,981, 7,399,590, 7,435,561,7,666,598, 7,763,427, 8,017,339, 8,030,000, 8,062,850, and 8,071,308,herein incorporated by reference in their entireties).

The recombinase of the reaction mixtures, compositions, and kitsincludes any suitable agent that can promote recombination betweenpolynucleotide molecules. The recombinase can be an enzyme thatcatalyzes homologous recombination. For example, the reaction mixturecan include a recombinase that includes, or is derived from, abacterial, eukaryotic or viral (e.g., phage) recombinase enzyme.

In any of the embodiments of the present teachings, a homologousrecombination enzyme is wild-type, mutant, recombinant, fusion, orfragments thereof. In some embodiments, a homologous recombinationenzyme includes an enzyme from any organism, including myoviridae (e.g.,uvsX from bacteriophage T4, RB69, and the like) Escherichia coli (e.g.,recA) or human (e.g., RAD51). In embodiments, the reaction mixtureincludes one or more recombinases selected from uvsX, RecA, RadA, RadB,Rad51, a homologue thereof, a functional analog thereof, or acombination thereof. In illustrative embodiments, the recombinase isuvsX. The UvsX protein can be present, for example, at 50-1000 ng/μl,100-750 ng/μl, 200-600 ng/μl, or 250 to 500 ng/μl.

In some embodiments, methods, kits, and compositions for nucleic acidamplification includes one or more recombinase accessory proteins in thepre-seeding reaction mixture. For example, an accessory protein canimprove the activity of a recombinase enzyme. In some embodiments, anaccessory protein can bind single strands of template nucleic acidmolecules or can load a recombinase onto a template nucleic acidmolecule. In some embodiments, an accessory protein is wild-type,mutant, recombinant, fusion, or fragments thereof. In some embodiments,accessory proteins can originate from any combination of the same ordifferent species as the recombinase enzymes that are used to conduct anucleic acid amplification reaction. Accessory proteins can originatefrom any bacteriophage including a myoviral phage. Examples of amyoviral phage include T4, T2, T6, Rb69, Aeh1, KVP40, Acinetobacterphage 133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4,cyanophage S-PM2, Rb14, Rb32, Aeromonas phage 25, Vibrio phage nt-1,phi-1, Rb16, Rb43, Phage 31, phage 44RR2.8t, Rb49, phage Rb3, and phageLZ2. Accessory proteins can originate from any bacterial species,including Escherichia coli, Sulfolobus (e.g., S. solfataricus) orMethanococcus (e.g., M. jannaschii). In some embodiments, methods fornucleic acid amplification can include single-stranded binding proteins.Single-stranded binding proteins include myoviral gp32 (e.g., T4 orRB69), Sso SSB from Sulfolobus solfataricus, MjA SSB from Methanococcusjannaschii, and E. coli SSB protein.

In some embodiments, methods for nucleic acid amplification includeproteins that improve recombinase loading onto a nucleic acid. Forexample, UvsY protein is a recombinase-loading protein. In someembodiments, the reaction mixture includes recombinase accessoryproteins. In illustrative embodiments, the recombinase accessory proteinis uvsY. UvsY can be present between about 20 and 500 ng/μl, forexample, between about 20 and 250 ng/μl, or between about 20 and 125ng/μl. In a non-limiting example, UvsY is present at between 75 and 125ng/μl.

In any of the embodiments of the present teachings, diffusion within thepre-seeding reaction mixture or the templating reaction mixture, islimited by the addition of a diffusion-limiting agent that is effectivein preventing or slowing the diffusion of one or more of thepolynucleotide templates and/or one or more of the amplificationreaction products through the pre-seeding reaction mixture. Inclusion ofa diffusion-limiting agent may be advantageous when amplifying two ormore template nucleic acid molecules within a single continuous liquidphase of a reaction mixture. For example, the diffusion-limiting agentcan prevent or slow diffusion of template nucleic acid molecules oramplified polynucleotides produced via replication of at least someportion of a template nucleic acid molecule within the pre-seedingreaction mixture, thus preventing the formation of polyclonalcontaminants without requiring compartmentalization of the pre-seedingreaction mixture by physical means or encapsulation means (e.g.,emulsions) during the amplification. Such methods of amplifyingtemplates within a single continuous liquid phase of a single reactionmixture without need for compartmentalization greatly reduces the cost,time, and effort associated with generation of libraries amenable forhigh-throughput methods such as digital PCR, next-generation sequencing,and the like.

In some embodiments, the diffusion-limiting agent is a sieving agent.The sieving agent can be any agent that is effective in sieving one ormore template nucleic acid molecules or polynucleotides present in thepre-seeding reaction mixture, such as for example amplification reactionproducts and/or template nucleic acid molecules. In some embodiments,the sieving agent restricts or slows the migration of polynucleotideamplification products through the pre-seeding reaction mixture. In someembodiments, the average pore size of the sieving agent is such thatmovement of a target component within the pre-seeding reaction mixture(e.g., a polynucleotide) is selectively retarded or prevented. In oneexample, the sieving agent includes any compound that provides a matrixhaving a plurality of pores that are small enough to slow or retard themovement of a polynucleotide or template nucleic acid molecule through areaction mixture containing the sieving agent. Thus, a sieving agent canreduce Brownian motion of a polynucleotide.

In some embodiments, the sieving agent is a polymer compound. In someembodiments, a sieving agent is a cross-linked or a non-cross linkedpolymer compounds. By way of non-limiting examples, the sieving agentcan include polysaccharides, polypeptides, organic polymers, or anyother suitable polymer. In any of the embodiments, a sieving agent ispolymers that are linear or branched. In some embodiments, a sievingagent is charged or neutral polymers. In some embodiments, the sievingagent can include a blend of one or more polymers, each having anaverage molecular weight and viscosity. In some embodiments, the sievingagent is a polymer with an average molecular weight of between about10,000 and 2,000,000 Da, for example between about 10,000 and 1,000,000Da, between about 10,000 and 500,000 Da, between about 10,000 and250,000 Da, or between about 10,000 and 100,000 Da. In certainembodiments, the polymer has an average molecular weight between about12,000 and 95,000 Da or between about 13,000 and 95,000 Da.

In some embodiments, a sieving agent exhibits an average viscosity rangeof about 5 centipoises to about 15,000 centipoises when dissolved inwater at 2 weight percent measured at about 25° C., or about 10centipoises to about 10,000 centipoises as a 2% aqueous solutionmeasured at about 25° C., or about 15 centipoises to about 5,000centipoise as a 2% aqueous solution measured at about 25° C.

In some embodiments, the sieving agent has a viscosity average molecularweight (M_(v)) of about 25 to about 1,5000 kM_(v), or about 75-1,000kM_(v), or about 85-800 kM_(v). In some embodiments, the reactionmixture includes a sieving agent at about 0.1 to about 20% weight pervolume (w/v), or about 1-10% w/v, or about 2-5% w/v.

In some embodiments, the sieving agent is a polysaccharide polymer. Insome embodiments, a sieving agent is a polymer of glucose or galactose.In some embodiments, a sieving agent is one or more of the followingpolymers: cellulose, dextran, starch, glycogen, agar, chitin, pectin, oragarose. In some embodiments, the sieving agent is a glucopyranosepolymer. In some embodiments, the sieving agent includes a cellulosederivative, such as sodium carboxy methyl cellulose, sodiumcarboxymethyl 2-hydroxyethyl cellulose, methyl cellulose, hydroxyl ethylcellulose, 2-hydroxypropyl cellulose, carboxy methyl cellulose, hydroxylpropyl cellulose, hydroxyethyl methyl cellulose, hydroxybutyl methylcellulose, (hydroxypropyl)methyl cellulose or hydroxyethyl ethylcellulose, or a mixture including any one or more of such polymers.

In some embodiments, the pre-seeding reaction mixture and/or thetemplating reaction mixture includes a mixture of different sievingagents, for example, a mixture of different cellulose derivatives,starch, polyacrylamide, and the like. In some embodiments, thepre-seeding reaction mixture includes a crowding agent. In someembodiments, the pre-seeding reaction mixture includes both a crowdingagent and a sieving agent.

In some embodiments, the diffusion-limiting agent is adiffusion-reducing agent. A diffusion-reducing agent includes anycompound that reduces migration of template nucleic acid molecules orpolynucleotides from a region of higher concentration to one having alower concentration. In some embodiments, a diffusion-reducing agentincludes any compound that reduces migration of any component of anucleic acid amplification reaction irrespective of size.

It should be noted that the concepts of a sieving agent and adiffusion-reducing agent are not necessarily mutually exclusive; asieving agent can frequently be effective in reducing diffusion oftarget compounds through a reaction mixture, whereas adiffusion-reducing agent can frequently have a sieving effect onreaction components. In some embodiments, the same compound orpre-seeding reaction mixture additive can act both as a sieving agentand/or a diffusion-reducing agent. Any of the sieving agents of thepresent teachings can in some embodiments be capable of acting as adiffusion-reducing agent and vice versa.

In some embodiments, the diffusion-reducing agent and/or sieving agentincludes polyacrylamide, agar, agarose or a cellulose polymer such ashydroxyethyl cellulose (HEC), methyl-cellulose (MC), or carboxymethylcellulose (CMC).

In some embodiments, the sieving agent and/or the diffusion-reducingagent is included in the pre-seeding reaction mixture at concentrationsof at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 90%, or95% w/v (weight of agent per unit volume of reaction mixture).

In some embodiments, the diffusion-limiting agent is a crowding agent.For example, a crowding agent can increase the concentration of one ormore components in a nucleic acid amplification reaction by generating acrowded reaction environment. In some embodiments, the pre-seedingreaction mixture includes both a sieving agent and/or diffusion reagentand a crowding agent.

In some embodiments, different template nucleic acid molecules arepre-seeded onto one or more different discrete supports (e.g., beads orparticles) without the need for compartmentalization prior toamplification. In other embodiments, the template nucleic acid moleculesare partitioned or distributed into emulsions prior to amplifying. Thepre-seeding reactions can be carried out in parallel in a plurality ofcompartmentalized reaction volumes, as opposed to amplification within asingle continuous liquid phase. Each reaction volume can include thepre-seeding reaction mixture. For example, the template nucleic acidmolecules can be distributed or deposited into an array of reactionchambers, or an array of reaction volumes, such that at least two suchchambers or volumes in the array receive a single template nucleic acidmolecule. In some embodiments, a plurality of separate reaction volumesis formed. The reaction chambers (or reaction volumes) can optionally besealed prior to amplification. Pre-seeding reactions can be performed ineach of the reaction chambers to generate substantially monoclonalpopulations of template nucleic acid molecules. In another embodiment,the reaction mixture is compartmentalized or separated into a pluralityof microreactors dispersed within a continuous phase of an emulsion.Each compartment or microreactor serves as an independent amplificationreactor, thus the entire emulsion is capable of supporting many separateamplification reactions in separate (discontinuous) liquid phases in asingle reaction vessel (e.g., an Eppendorf tube or a well). As usedherein, the term “emulsion” includes any composition including a mixtureof a first liquid and a second liquid, wherein the first and secondliquids are substantially immiscible with each other. Thecompartmentalized or separate reaction volumes optionally do not mix orcommunicate, or are not capable of mixing or communicating, with eachother. The pre-seeding reaction mixtures in the microreactors can be anyof the pre-seeding reaction mixtures discussed herein.

In some embodiments, the nucleic acid synthesis method further includesrecovering from the emulsion at least some of the supports attached tosubstantially monoclonal populations of template nucleic acid molecules.In some embodiments, the nucleic acid synthesis method further includesdepositing onto a surface at least some of the supports attached to thesubstantially monoclonal populations of template nucleic acid molecules.In some embodiments, the nucleic acid synthesis method further includesforming an array by depositing onto a surface at least some of thesupports attached to the substantially monoclonal populations oftemplate nucleic acid molecules.

In some embodiments, the disclosure relates generally to compositions,as well as systems, methods, kits and apparatuses, which includes thepre-seeding reaction mixtures and templating reaction mixtures.Accordingly, provided herein in certain embodiments are reactionmixtures that include a polymerase and one or more pre-seeded supports.The reaction mixture compositions can include a recombinase andoptionally a recombinase accessory protein as known for an RPA reaction.In such embodiments, the reaction mixture, especially the pre-seedingreaction mixtures, can include between 5×10⁷ and 10⁹ template nucleicacid molecules in a solution that includes between 5×10⁶ and 10¹⁰ beads,such as Ion Sphere Particles. In illustrative examples, 1 or less than 1template nucleic acid molecule is included per bead in the pre-seedingreaction mixture. For example, between about 0.1 and 0.9 templatenucleic acid molecules can be included per solid support, for examplebetween about 0.1 and 0.7 template nucleic acid molecules, between about0.1 and 0.5 template nucleic acid molecules, or between about 0.1 and0.3 template nucleic acid molecules can be included per solid support.The templating reaction mixtures in certain examples, include less than1,000,000, 500,000, 1,000, 500, 100, 10, or 0 template nucleic acidmolecules in solution along with pre-seeded solid supports of thepresent teachings. In the pre-seeding and the templating reactionmixtures, the polymerase and optionally the recombinase are typicallypresent at effective concentrations for amplification, such as known foran RPA reaction, or at higher concentrations such that they can becombined with other reaction components into a final pre-seedingreaction mixture. In any of the embodiments of the present teachings thevolumes of the pre-seeding reaction mixtures and/or templating reactionmixtures are between about 50 and 2,000 μl, for example between about 50and 1,500 μl, between about 50 and 1,000 μl, between about 50 and 500μl, between about 50 and 250 μl, or between about 50 and 150 μl. In anyof the disclosed embodiments, the volume of the pre-seeding reaction andthe volume of the templating reaction mixture is different.

The pre-seeding reaction mixtures, and templating reaction mixtures, canfurther include other components. For example, the compositions caninclude nucleotides, a population of first primers, optionally a secondprimer, cofactors, and a buffer. The population of first primers andoptionally a population of second primers can be attached to the one ormore supports. As a non-limiting example, the composition include one ormore supports, a recombinase such as uvsX, a polymerase such as Sau DNApolymerase, a recombinase-loading protein such as uvsY, asingle-stranded binding protein such as gp32 protein, nucleotides, ATP,phosphocreatine, and creatine kinase. The composition can be in liquidform, or it can be in a solid form, such as a dried-down pellet formthat can be rehydrated. Furthermore, components of the compositions, canbe split up such that any combination of the components can be in apellet or liquid form, and one or more combinations of the rest of thecomponents can be in one or more separate pellet or liquid forms. Suchcombinations can form kits that include at least two of suchcombinations. For example, a kit can include a pellet that includes allthe reaction mixture components of the present teachings except for thepolymerase enzyme, which can be provided in a separate pellet or liquidin the kit.

In illustrative embodiments, a composition includes a population oftemplate nucleic acid molecules, a polymerase, a recombinase, a forwardprimer, a reverse primer, nucleotides, and a buffer. In someembodiments, the composition includes a template nucleic acid molecule,a forward primer, a reverse primer, uvsX recombinase, uvsY recombinaseloading protein, gp32 protein, Sau DNA polymerase, dNTPs, ATP,phosphocreatine, and creatine kinase.

In some embodiments, a composition includes at least two differenttemplate nucleic acid molecules with both a first primer bindingsequence and a second primer binding sequence, a recombinase, arecombinase accessory protein, a polymerase, a first universal primer, asecond universal primer, dNTPs, and a buffer. In some embodiments, thecomposition further includes one or more supports. In illustrativeembodiments, the composition includes at least two different templatenucleic acid molecules with both a first primer binding sequence and asecond primer binding sequence, uvsX recombinase, uvsY recombinaseloading protein, gp32 protein, Sau DNA polymerase, ATP, phosphocreatine,creatine kinase, a first universal primer attached to a bead support, asecond universal primer, and a buffer.

In some embodiments, the pre-seeding reaction mixture or the templatingreaction mixture is formed by the individual addition of each componentto an aqueous or emulsion solution. In other embodiments, the reactionmixture is in the form of a dehydrated pellet that requires rehydrationprior to use. The dehydrated pellet can include, for example,recombinase, an optional recombinase accessory proteins, optionallygp32, DNA polymerase, dNTPs, ATP, optionally phosphocreatine, anoptional crowding agent, and optionally creatine kinase. Rehydrationbuffer can include, for example, Tris buffer, potassium acetate salt,and optionally a crowding agent such as PEG. The DNA polymerase, can befor example, T4 or T7 DNA polymerase, and can further includethioredoxin when the polymerase is T7 DNA polymerase. In someembodiments, when a dehydrated pellet is used that includes reactionmixture components, the pellet is rehydrated with a rehydration bufferand template nucleic acid molecules, primers, and additionalnuclease-free water are added to a final volume.

In some embodiments, the pre-seeding reaction mixture or the templatingreaction mixture is pre-incubated under conditions that inhibitpremature reaction initiation. For example, one or more components inthe pre-seeding reaction mixture can be withheld from a reaction vesselto prevent premature reaction initiation. To start the reaction, adivalent cation is added (e.g., magnesium or manganese). In anotherexample, the pre-seeding reaction mixture is pre-incubated at atemperature that inhibits enzyme activity. The reaction can bepre-incubated at about 0-15° C. or about 15-25° C. to inhibit prematurereaction initiation. The reaction is then incubated at a highertemperature to increase enzymatic activity. In illustrative embodiments,the pre-seeding reaction mixture is not exposed to a temperature above42° C. during the reaction.

In any of the disclosed embodiments, the pre-seeding reaction optionallyincludes repeated cycles of nucleic acid amplification. A cycle ofamplification optionally includes (a) hybridization of a first primer toa template strand, (b) primer extension to form a first extended strand,(c) partial or incomplete denaturation of the extended strand from thetemplate strand. Optionally, the denatured portion of the templatestrand from step (c) is free to hybridize with a different first primerin the next amplification cycle. In some embodiments, primer extensionin a subsequent amplification cycle involves displacement of the firstextended strand from the template strand. A second primer can beincluded which hybridizes to the 3′ end of the first extended strand. Insome embodiments, the disclosed methods (and related compositions,systems, and kits) further include one or more primer extension steps.For example, the methods include extending a primer via nucleotideincorporation using a polymerase. In embodiments, extending a primerincludes contacting the hybridized primer with a polymerase and one ormore types of nucleotides under nucleotide incorporation conditions.Typically, extending a primer occurs in a template-dependent fashion.Optionally, the disclosed methods (and related compositions, systems,and kits) include extending the first primer by incorporating one ormore nucleotides onto the 3′ OH of the first primer of the firstprimer-template duplex using the polymerase, thereby forming an extendedfirst primer. Optionally, the disclosed methods (and relatedcompositions, systems, and kits) include binding a second primer to thesecond primer binding sequence of the first extended primer by anysuitable method (e.g., ligation or hybridization). Optionally, thedisclosed methods (and related compositions, systems, and kits) includeextending the second primer by incorporating one or more nucleotidesinto the second primer of the second primer-template duplex using thepolymerase, thereby forming an extended second primer.

In some embodiments, extending the first primer results in formation ofa first extended primer. The first extended primer can include some orall of the sequence of the reverse strand of the template. Optionally,the first extended primer includes a second primer binding sequence. Insome embodiments, extending the second primer results in formation of asecond extended primer. The second extended primer can include some orall of the sequence of the forward strand of the template. Optionally,the second extended primer includes a first primer binding sequence. Insome embodiments, the methods (and related compositions, systems, andkits) can further include attaching one or more extended primer strandsto a support. The attaching can optionally be performed during theamplifying or alternatively after the amplification is complete. In someembodiments, the support is attached to a population of first primers.For example, the support can include a population of first primers, andthe methods can include hybridizing at least one of the extended secondprimers to a first primer of the support, thereby attaching the extendedsecond primer to the support. For example, the first primer canhybridize to a first primer binding sequence in the extended secondprimer. In some embodiments, the support includes multiple instances ofa second primer and the methods include hybridizing at least one of theextended first primer strands to a second primer of the support, as inbridge PCR.

In any of the embodiments of the present teachings, the pre-seedingreaction is carried out using an RPA reaction where partial denaturationand/or amplification, including any one or more steps or methodsdescribed herein, is achieved using a polymerase, recombinase, andtypically a recombinase accessory protein. Not to be limited by theory,it is believed that the recombinase coats single-stranded DNA (ssDNA) toform a nucleoprotein filament strand which invades a double-strandedregion of homology on template nucleic acid molecules. This creates ashort hybrid and a displaced strand bubble known as a D-loop. The free3′-end of the hybridized primer is extended by DNA polymerases tosynthesize a new complementary strand. The complementary stranddisplaces the originally-paired partner strand of the template nucleicacid molecule as it is elongated. In an embodiment, one or more of apair of primers are contacted with one or more recombinases before beingcontacted with a template nucleic acid molecule, which is optionallydouble-stranded. RPA reactions are typically isothermal and is performedwithin an emulsion.

In any of the embodiments of the present teachings, the pre-seedingreaction is carried out by template walking where portions ofdouble-stranded nucleic acid molecules become dissociated such that aprimer is bound to one of the strands to initiate a new round ofreplication (see, for example, U.S. Patent Publ. No. 2012/0156728,published Jun. 21, 2012, incorporated by reference herein in itsentirety). An embodiment of template walking includes a method of primerextension, including: (a) a primer-hybridization step, (b) an extensionstep, and (c) a walking step. Optionally, the primer-hybridization stepincludes hybridizing a first primer to a first primer binding sequenceon a template nucleic acid molecule (“reverse strand”). Optionally theextension step includes generating an extended first forward strand thatis a full-length complement of the reverse strand and is hybridizedthereto. The extended first forward strand is, for example, generated byextending the first forward primer in template-dependent fashion usingthe reverse strand as template. Optionally the walking step includeshybridizing another first primer to the first primer binding sequencewhere the reverse strand is also hybridized to the first forward strand.For example, the walking step includes denaturing at least a portion ofthe first primer binding sequence from the forward strand, where anotherportion of the reverse strand remains hybridized to the forward strand.In any of the disclosed embodiments, the reaction mixture includes oneor more supports with the first primer bound thereto, wherein the firstprimer binding sequence on at least one of the template nucleic acidmolecules is complementary or identical to at least a portion of thefirst primer. In some embodiments, at least one of the template nucleicacid molecules have a second primer binding sequence that iscomplementary or identical to at least a portion of a second primer. Insome embodiments, the second primer is also bound to the support suchthat amplification can occur back and forth on the surface. In variousembodiments, the second primer is in solution. In other embodiments, thesecond primer is immobilized on the support. Template walking reactionsare typically performed at isothermal temperatures and is performedwithin an emulsion.

Template walking can result in the introduction of many identicalnucleotides at the proximal terminus of the template nucleic acidmolecule attached to the support. In some embodiments, template walkingis performed on one or more supports in such a way that the attachedtemplate nucleic acid molecules have fewer than 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, or300 identical nucleotides introduced at the proximal terminus of thetemplate nucleic acid molecule attached to the support. In otherembodiments, template walking is performed on one or more supports insuch a way that the attached template nucleic acid molecules havebetween about 10 and 300 identical nucleotides introduced at theproximal terminus of the template nucleic acid molecule attached to thesupport, for example between about 10 and 200 identical nucleotides,between about 10 and 150, or between about 10 and 100 identicalnucleotides introduced at the proximal terminus of the template nucleicacid molecule attached to the support. In any embodiment, the proximalterminus is the terminus of the template nucleic acid molecule closestto the support to which each template nucleic acid molecule is attached.In some aspects, a template nucleic acid molecule attached to thesupport is a series of identical nucleotides attached to the templatenucleic acid molecule or template nucleic acid segment. In someembodiments, less than 5%, 10%, 15%, 20%, or 25% of each of thesubstantially monoclonal populations of template nucleic acid moleculeshave variable numbers of an identical nucleotide at the proximalterminus. For example, less than 5%, 10%, 15%, 20%, or 25% of the eachof the substantially monoclonal populations of template nucleic acidmolecules can have between about 10 and 300 identical nucleotidesintroduced at the proximal terminus of the template nucleic acidmolecule attached to the support, for example between about 10 and 200identical nucleotides, between about 10 and 150, between about 10 and100, or between about 20 and 50 identical nucleotides introduced at theproximal terminus of the template nucleic acid molecule attached to thesupport.

In any of the disclosed embodiments, the pre-seeding reaction is carriedout using PCR methods. A skilled artisan will recognize various methodsto perform PCR that will generate substantially monoclonal populationsof template nucleic acid molecules. In some embodiments, the pre-seedingreaction is carried out in a single round of PCR. In other embodiments,the pre-seeding reaction is carried out in multiple rounds of PCR. Forexample, the methods can include diluting the amount of template nucleicacid molecules that are reacted with the supports to reduce thepercentage of supports that react with more than one template nucleicacid molecule. In some embodiments, the template nucleic acid moleculesare diluted such that the pre-seeding reactions have asupport-to-template nucleic acid molecule ratio that is selected tooptimize the percentage of supports having a substantially monoclonalpopulation of template nucleic acid molecules attached thereto. Forexample, the pre-seeding reaction can be carried out with asupport-to-template nucleic acid molecule ratio of at least about 1:1,1.25:1, 1.5:1, 1.75:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1 10:1,25:1, 50:1, 75:1, and 100:1. In some embodiments, PCR is performed in anemulsion where the PCR is carried out in a plurality of microreactors inan emulsion as described elsewhere herein.

The described methods for pre-seeding supports include immobilizing oneor more reaction components (for example, one or more template nucleicacid molecules and/or primers) during the pre-seeding reaction toprevent cross contamination of amplification reaction products andconsequent reduction in monoclonality. One such example includes bridgeamplification, where all of the primers required for amplification(e.g., forward and reverse primer) are attached to the surface of amatrix support. In addition to such immobilization, additionalimmobilization components are included in the reaction mixture. Forexample, the template nucleic acid molecule and/or amplification primerscan be suspended in gels or other matrices during the amplification soas to prevent migration of amplification reaction products from the siteof synthesis. Such gels and matrices typically require to be removedsubsequently, requiring the use of appropriate “melting” or otherrecovery steps and consequent loss of yield.

In any of the embodiments of the present teachings, the pre-seedingreaction is carried out under isothermal conditions. In someembodiments, isothermal conditions include a reaction subjected to atemperature variation which is constrained within a limited range duringat least some portion of the amplification (or the entire amplificationprocess), including for example a temperature variation that is equal orless than about 10° C., or about 5° C., or about 1-5° C., or about0.1-1° C., or less than about 0.1° C., or, for example a temperaturevariation is equal or less than or 10° C., or 5° C., or 1-5° C., or0.1-1° C., or less than 0.1° C. The temperature of the isothermalreaction can be typically between about 15° C. and 65° C., for examplebetween about 15° C. and 55° C., between about 15° C. and 45° C.,between about 15° C. and 37° C., between about 30° C. and 60° C.,between about 40° C. and 60° C., between about 55° C. and 60° C.,between about 35° C. and 45° C., or between about 37° C. and 42° C. Inother embodiments, the pre-seeding reaction is not exposed to atemperature above 40° C., 41° C., 42° C., 43° C., 45° C., or 50° C.Accordingly, in certain embodiments, the reaction mixture is not exposedto hot start conditions. However, it is understood the enzymes used atthese temperatures will need to be optimized in combination and mayrequire changes in the enzyme, for example, using a different DNApolymerase, such as Bst instead of Bsu. A rate limiting enzyme may bethe polymerase, wherein a high concentration or excess (i.e.non-limiting) amount of the polymerase or a lower temperature ensuresthe amplification reaction proceeds based on the kinetics of thepolymerase.

The pre-seeding reaction can be performed for 0.25 minutes to 240minutes, thereby amplifying the nucleic acid template. In certainembodiments, the pre-seeding reaction is performed for between about0.25 and 240 minutes, for example between about 0.25 and 120 minutes,between about 0.25 and 60 minutes, between about 0.25 and 30 minutes,between about 0.25 and 15 minutes between about 0.25 and 10 minutes,between about 0.25 and 7.5 minutes, between about 0.25 and 5 minutes, orbetween about 2 and 5 minutes. In further illustrative embodiments, thepre-seeding reaction is performed for between about 1.5 and 10 minutes,for example between about 1.5 and 8 minutes, between about 1.5 and 6minutes, between about 1.5 and 5 minutes, or between about 1.5 and 4minutes, the reaction is isothermal, and the temperature of the reactionis between about 35° C. and 65° C., for example between about 35° C. and55° C., between about 35° C. and 45° C., between about 30° C. and 60°C., between about 40° C. and 60° C., between about 40° C. and 55° C.,between about 50° C. and 60° C., or between about 37° C. and 42° C. Forexample, the pre-seeding reaction can be between performed for betweenabout 1 and 10 minutes in an isothermal reaction where the temperatureof the reaction can be between about 35° C. and 45° C. or thepre-seeding reaction can be between performed for between about 2 and 5minutes in an isothermal reaction where the temperature of the reactioncan be between about 37° C. and 42° C.

In some embodiments, the methods are performed without subjecting thedouble-stranded template nucleic acid molecules to extreme denaturingconditions during the amplifying. For example, the methods can beperformed without subjecting the template nucleic acid template(s) totemperatures equal to or greater than the T_(m) of the template(s)during the amplifying. In some embodiments, the methods are performedwithout contacting the template(s) with chemical denaturants such asNaOH, urea, guanidium, and the like, during the amplifying. In someembodiments, the amplifying includes isothermally amplifying.

In some embodiments, the methods are performed without subjecting thetemplate nucleic acid molecules to extreme denaturing conditions duringbetween about 2 and 50 consecutive cycles, between about 2 and 40consecutive cycles, between about 2 and 30 consecutive cycles, betweenabout 2 and 25 consecutive cycles, between about 2 and 20 consecutivecycles, or between about 2 and 15 consecutive cycles. For example, themethods can include between about 2 and 50 consecutive cycles, betweenabout 2 and 40 consecutive cycles, between about 2 and 30 consecutivecycles, between about 2 and 25 consecutive cycles, between about 2 and20 consecutive cycles, or between about 2 and 15 consecutive cycles ofnucleic acid synthesis without contacting the nucleic acid template(s)with a chemical denaturant or raising the temperature above 50° C. or55° C. In some embodiments, the methods include performing between about2 and 50 consecutive cycles, between about 2 and 40 consecutive cycles,between about 2 and 30 consecutive cycles, between about 2 and 25consecutive cycles, between about 2 and 20 consecutive cycles, orbetween about 2 and 15 consecutive cycles of nucleic acid synthesiswithout subjecting the nucleic acid template(s) to temperatures that aregreater than 25° C., 20° C., 15° C., 10° C., 5° C., 2° C. or 1° C. belowthe actual or calculated T_(m) of the template or population oftemplates (or the actual or calculated average T_(m) of the template orpopulation of templates). The consecutive cycles of nucleic acidsynthesis may or may not include intervening steps of partialdenaturation and/or primer extension. Optionally, the at least one cycleof template-based replication includes a partial denaturation step, anannealing step, and an extension step. In some embodiments, the attachedidentical primers in the pre-seeding reaction is a sequence ofadenosines or uridines or some combination of adenosines and uridinesbetween about 5 and 100 nucleotides in length, for example between about5 and 80 nucleotides in length, between about 5 and 60 nucleotides inlength, between about 5 and 40 nucleotides in length, between about 10and 75 nucleotides in length, between about 15 and 75 nucleotides inlength, or between about 20 and 50 nucleotides in length.

The pre-seeding reaction result in the formation of a population ofpre-seeded supports with populations of substantially monoclonaltemplate nucleic acid molecules attached thereto. In various aspects,the substantially monoclonal populations of template nucleic acidmolecules are template nucleic acid molecules with at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%or 100% identity at the sequence level. In some embodiments, thepercentage of substantially monoclonal template nucleic acid moleculesattached to a pre-seeded support are 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the template nucleic acid molecules attachedthereto.

In various embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% of the supports in a population of pre-seeded supports havesubstantially monoclonal populations of nucleic acid molecules attachedduring the pre-seeding reaction. In illustrative embodiments, at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%of the supports in a population of pre-seeded supports havesubstantially monoclonal populations of nucleic acid molecules attachedduring the pre-seeding reaction.

In some embodiments, a pre-seeding reaction generate supports havingzero template nucleic acid molecules attached thereto (empty solidsupports), other pre-seeded supports having one type of template nucleicacid molecule attached thereto, and other pre-seeded supports havingmore than one type of template nucleic acid molecule attached thereto.In any of the embodiments of the present teachings, the number ofattached template nucleic acid molecules in the populations ofsubstantially monoclonal template nucleic acid molecules attached to oneor more pre-seeded supports are the pre-seeding number. In any of thedisclosed embodiments, the pre-seeding number is between about 1 and150,000 template nucleic acid molecules, for example between about 1 and100,000, between about 1 and 75,000, between about 1 and 50,000, betweenabout 1 and 25,000, between about 1 and 10,000, between about 1 and5,000, or between about 1 and 2,500 template nucleic acid molecules. Inillustrative embodiments, the pre-seeding number is between about 10 and100,000 template nucleic acid molecules, for example between about 10and 75,000, between about 10 and 50,000, between about 10 and 25,000,between about 10 and 10,000, between about 10 and 5,000, or betweenabout 10 and 2,500 template nucleic acid molecules.

In some embodiments, after the pre-seeding reaction, a majority of anyprimers attached to a support are not bound to a template nucleic acidmolecule. These unbound primers can be used in the subsequent templatingreaction for further amplification of the template nucleic acidmolecules. For example, after the pre-seeding reaction, at least 90%,95%, 96%, 97%, 98%, or 99% of primers attached to a support aretypically not bound to a template nucleic acid molecule.

In some embodiments, the disclosure relates generally to methods, aswell as systems, compositions, kits and apparatuses, wherein the methodstypically include a templating reaction after the pre-seeding reactionwherein the template nucleic acid molecules on the pre-seeded supportsare further amplified (herein referred to as the templating reaction).The templating reaction mixture does not include additional templatenucleic acid molecules in solution such that the template nucleic acidmolecules attached to the one or more pre-seeded supports are thepredominant or only source of template nucleic acid molecules in thetemplating reaction mixture before the templating reaction is initiated.In illustrative embodiments, one or more washes are carried out on theone or more pre-seeded supports before introducing them into thetemplating reaction mixture. In some embodiments, less than 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1% of the template nucleic acidmolecules in solution at the end of the pre-seeding reaction mixture arepresent in the templating reaction. In any of the embodiments of thepresent teachings, the percentage of template nucleic acid molecules inthe templating reaction mixture that are attached (have been pre-seeded)on one or more supports before the templating reaction is initiated areat least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, or 99.9% of the template nucleic acid molecules in thereaction mixture.

In any of the embodiments of the present teachings, the templatingreaction is typically an RPA reaction. As such, any details regardingcomponents and conditions for an RPA reaction discussed above for apre-seeding reaction apply to the templating reaction except thattemplate nucleic acids are typically present in the initial reactionmixture for a pre-seeding reaction, but not for a templating reaction.The templating reaction mixture typically includes all or some of thefollowing: one or more pre-seeded solid supports that include apopulation of attached substantially identical first primers and havesubstantially monoclonal template nucleic acid molecules attachedthereto, a polymerase, a recombinase, an optional single-strandedbinding protein, an optional recombinase loading protein, an optionalsecond or reverse primer, which can be attached to the solid support,but in illustrative examples, is in solution, dNTPs, ATP, a buffer, andoptionally one or both of phosphocreatine and creatine kinase. Adivalent cation, such as MgCl₂ or Mg(OAc)₂, can be added to start thereaction. In various embodiments, the buffer included a crowding agent,such as PEG, Tris buffer, and/or a potassium acetate salt. A forwardprimer binding sequence on template nucleic acid molecules iscomplementary or identical to at least a portion of the forward primerand the optional reverse primer binding sequence on the template nucleicacid molecules is complementary or identical to at least a portion ofthe reverse primer. Reaction mixtures for templating reactionsthemselves form separate aspects of the invention. In furtherillustrative embodiments, the RPA templating reaction is a bulkisothermal amplification or is performed in wells of a multi-well solidsupport (e.g. see the pre-seeding amplification reaction discussedfurther herein).

In some embodiments, the templating reaction mixture is pre-incubatedunder conditions that inhibit premature reaction initiation. Forexample, one or more components in the templating reaction mixture canbe withheld from a reaction vessel to prevent premature reactioninitiation. To start the reaction, a divalent cation is added (e.g.,magnesium or manganese). In another example, the templating reactionmixture is pre-incubated at a temperature that inhibits enzyme activity.The reaction is pre-incubated at about 0-15° C. or about 15-25° C. toinhibit premature reaction initiation. The reaction can then beincubated at a higher temperature to increase enzymatic activity. Inillustrative embodiments, the templating reaction mixture is not exposedto a temperature above 42° C. during the reaction.

Since the templating reaction is typically an RPA reaction, it iscarried out under isothermal conditions. In some embodiments, isothermalconditions include a reaction subjected to a temperature variation whichis constrained within a limited range during at least some portion ofthe amplification (or the entire amplification process), including forexample a temperature variation that is equal or less than about 10° C.,or about 5° C., or about 1-5° C., or about 0.1-1° C., or less than about0.1° C., or, for example a temperature variation is equal or less thanor 10° C., or 5° C., or 1-5° C., or 0.1-1° C., or less than 0.1° C. Thetemperature of the isothermal reaction is typically between about 15° C.and 65° C., for example between about 15° C. and 55° C., between about15° C. and 45° C., between about 15° C. and 37° C., between about 30° C.and 60° C., between about 40° C. and 60° C., between about 55° C. and60° C., between about 35° C. and 45° C., or between about 37° C. and 42°C. about 15° C. In other embodiments, the pre-seeding reaction is notexposed to a temperature above 40° C., 41° C., 42° C., 43° C., 45° C.,or 50° C. Accordingly, in certain embodiments, the reaction mixture isnot exposed to hot start conditions. However, it is understood theenzymes used at these temperatures will need to be optimized incombination and may require changes in the enzyme, for example, using adifferent DNA polymerase, such as Bst instead of Bsu. A rate limitingenzyme may be the polymerase, wherein a high concentration or excess(i.e. non-limiting) amount of the polymerase or a lower temperatureensures the amplification reaction proceeds based on the kinetics of thepolymerase.

In any of embodiments of the present teachings, the templating reactionis typically an RPA reaction that is performed for between about 0.25and 240 minutes, for example between about 0.25 and 120 minutes, betweenabout 0.25 and 60 minutes, between about 0.25 and 30 minutes, betweenabout 0.25 and 15 minutes between about 0.25 and 10 minutes, betweenabout 0.25 and 7.5 minutes, between about 0.25 and 5 minutes, or betweenabout 2 and 5 minutes. In illustrative embodiments, the templatingreaction is performed for between about 10 and 120 minutes, for examplebetween about 10 and 60 minutes, between about 10 and 45 minutes,between about 10 and 30 minutes, or between about 10 and 20 minutes. Infurther illustrative embodiments, the templating reaction is performedfor between about 20 and 60 minutes, for example between about 20 and 50minutes, between about 20 and 40 minutes, between about 20 and 35minutes, or between about 20 and 30 minutes.

In any of the embodiments of the present teachings, the templatingreaction includes amplifying a population of different template nucleicacid molecules on one or more pre-seeded supports to generate one ormore templated supports. For example, after the templating reaction,there can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 100, 250, 500,1,000, 2,500, 5,000, 10,000, 25,000, 50,000, 100,000, 250,000, 500,000,or 10⁶ times as many attached substantially monoclonal template nucleicacid molecules present on the templated solid supports as were presenton the pre-seeded solid supports. In illustrative embodiments, there isat least 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 25,000,or 50,000 times as many attached substantially monoclonal templatenucleic acid molecules present on the templated solid supports as werepresent on the pre-seeded solid supports. In some embodiments, at least50,000, 75,000 or 100,000 substantially monoclonal template nucleic acidmolecules or between about 25,000 and 1,000,000 substantially monoclonaltemplate nucleic acid molecules are present on the pre-seeded solidsupports, for example between about 25,000 and 500,000, between about25,000 and 250,000, between about 25,000 and 125,000, or between about25,000 and 100,000 substantially monoclonal template nucleic acidmolecules are present on the pre-seeded solid supports. The support orsupports typically remain in fluid communication during the templatingreaction.

Since the pre-seeding reaction can be an RPA reaction and the templatingreaction is typically an RPA reaction according to the presentteachings, the methods include sequential RPA reactions. A first RPAreaction is a pre-seeding reaction, followed by a second RPA templatingreaction. The reactions are carried out under the same conditions.However, in illustrative embodiments, the pre-seeding RPA reaction iscarried out such that less amplification cycles occur, than for thetemplating RPA reaction. For example, a pre-seeding RPA reaction can becarried out for less time than a templating RPA reaction that amplifiestemplate nucleic acid molecules attached to pre-seeded solid supportsgenerated by the pre-seeded RPA reaction. As non-limiting illustrativeexamples, a pre-seeding RPA reaction is carried out for 2 to 5 minutesto generate one or more, for example a population of pre-seeded solidsupports, which are then subjected to a templating RPA reaction that iscarried out for between 10 and 60 minutes. In these non-limitingillustrative examples, reaction components including template nucleicacids are washed away from the pre-seeded solid supports before thetemplating RPA reaction. All other reaction components of thepre-seeding and templating reaction, except for template nucleic acidmolecules in solution, are included in both the pre-seeding andtemplating reactions.

In some embodiments, after the templating reaction the percentage ofsites on a support or supports in a population of supports withsubstantially monoclonal template nucleic acid molecules attachedthereto are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of thetotal amplified supports (i.e., total supports include empty, polyclonalor substantially monoclonal populations of template nucleic acidmolecules). In illustrative embodiments, after the templating reactionthe percentage of sites on a support or supports in a population ofsupports with substantially monoclonal template nucleic acid moleculesattached thereto are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% of the total amplified supports (i.e.,total supports include empty, polyclonal or substantially monoclonalpopulations of template nucleic acid molecules) recovered from thereaction mixture. In some embodiments, after the templating reaction thepercentage of sites on a support or supports in a population of supportswith substantially monoclonal template nucleic acid molecules attachedthereto are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of thetotal supports recovered from the reaction mixture (i.e., total supportsincluding supports with no attached template nucleic acid molecules andsupports with either polyclonal or substantially monoclonal populationof template nucleic acid molecules). In illustrative embodiments, afterthe templating reaction the percentage of sites on a support or supportsin a population of supports with substantially monoclonal templatenucleic acid molecules attached thereto are at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the totalsupports recovered from the reaction mixture (i.e., total supportsincluding supports with no attached template nucleic acid molecules andsupports with either polyclonal or substantially monoclonal populationof template nucleic acid molecules).

In some embodiments, at least a portion of a primer hybridize with aportion of at least one strand of a polynucleotide in the templatingreaction mixture. For example, at least a portion of a primer canhybridize with a nucleic acid adapter that is joined to one or both endsof the polynucleotide. In some embodiments, at least a portion of aprimer is partially or fully complementary to a portion of thepolynucleotide or to the nucleic acid adapter. In some embodiments, aprimer is compatible for use in any type of sequencing platformincluding chemical degradation, chain-termination,sequencing-by-synthesis, pyrophosphate, massively parallel,ion-sensitive, and single-molecule platforms.

In some embodiments, a primer (e.g., first, second or third primer) hasa 5′ or 3′ overhang tail (tailed primer) that does not hybridize with aportion of at least one strand of a polynucleotide in the reactionmixture. In some embodiments, a tailed primer is any length, includingbetween about 1 and 100 nucleotides in length, for example between about1 and 90, between about 1 and 80 nucleotides in length, between about 1and 70 nucleotides in length, between about 1 and 60 nucleotides inlength, between about 1 and 50 nucleotides in length, between about 1and 40 nucleotides in length, or between about 1 and 30 nucleotides inlength.

The disclosed methods result in the production of a population ofamplicons, at least some of which amplicons in certain embodiments,include an amplified nucleic acid population. The amplified populationsproduced by the methods of the disclosure are useful for a variety ofpurposes. In some embodiments, the disclosed methods (and relatedcompositions, systems, and kits) optionally include further analysisand/or manipulation of the amplified populations (amplicons). Forexample, in some embodiments, the numbers of amplicons exhibitingcertain desired characteristics are detected and optionally quantified.In some embodiments, the amplifying is followed by sequencing theamplified product. The amplified product that is sequenced can be anamplicon that is a substantially monoclonal nucleic acid population. Insome embodiments, the disclosed methods include amplifying singlemembers of a population of amplicons at different sites or on differentsupports. The different sites optionally form part of an array of sites.In some embodiments, the sites in the array of sites include wells(reaction chambers) on the surface of an isFET array. Optionally, thenucleic acid molecule to be sequenced is positioned at a site. The sitecan include a reaction chamber or well. The site can be part of an arrayof similar or identical sites. The array can include a two-dimensionalarray of sites on a surface (e.g., of a flowcell, electronic device,transistor chip, reaction chamber, channel, and the like), or athree-dimensional array of sites within a matrix or other medium (e.g.,solid, semi-solid, liquid, fluid, and the like). In any of theembodiments of the present teachings, the site is operatively coupled toa sensor. The method includes detecting the nucleotide incorporationusing the sensor. Optionally, the site and the sensor are located in anarray of sites coupled to sensors.

In any of the embodiments of the present teachings, after the templatingreaction the templated supports have at least 50,000, 75,000, 100,000,125,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000,or 500,000 substantially monoclonal template nucleic acid moleculesattached to each templated support. In some embodiments, after thetemplating reaction the templated supports have between about 50,000 and500,000 substantially monoclonal template nucleic acid moleculesattached to each templated support, for example between about 50,000 and400,000 substantially monoclonal template nucleic acid molecules,between about 50,000 and 300,000 substantially monoclonal templatenucleic acid molecules, between about 50,000 and 200,000 substantiallymonoclonal template nucleic acid molecules, or between about 50,000 and100,000 substantially monoclonal template nucleic acid moleculesattached to each templated support. In illustrative embodiments, afterthe templating reaction the templated supports have between about100,000 and 400,000 substantially monoclonal template nucleic acidmolecules attached to each templated support, between about 100,000 and300,000 substantially monoclonal template nucleic acid molecules,between about 100,000 and 200,000 substantially monoclonal templatenucleic acid molecules, or between about 150,000 and 300,000substantially monoclonal template nucleic acid molecules attached toeach templated support.

In any of the disclosed embodiments, the amplified template nucleic acidmolecules on the supports is sequenced. Sequencing methods can includeany suitable method of sequencing known in the art. In some embodiments,template nucleic acid molecules that have been amplified according tothe present teachings are used in any nucleic acid sequencing workflow,including sequencing by oligonucleotide probe ligation and detection(e.g., SOLiD™ from Life Technologies, WO 2006/084131), probe-anchorligation sequencing (e.g., Complete Genomics™ or Polonator™),sequencing-by-synthesis (e.g., Genetic Analyzer and HiSeg™, fromIllumina), pyrophosphate sequencing (e.g., Genome Sequencer FLX from 454Life Sciences), ion-sensitive sequencing (e.g., Personal Genome Machine(PGM™), Ion Proton™ Sequencer, Ion S5, and Ion S5 XL, all from IonTorrent™ Systems, Inc.), single molecule sequencing platforms (e.g.,HeliScope™ from Helicos™) nanopore sequencing via read of individualbases as they pass through the nanopores (e.g. MinION from OxfordNanopore Technologies), chemical degradation sequencing, capillaryelectrophoresis, gel electrophoresis, and any other next-generation,massively parallel sequencing platforms.

In some sequencing devices, e.g., Ion Torrent Systems, sequencingreactions are conducted in microwells of a surface such as, for example,a semiconductor chip. An exemplary embodiment of the use of a beadsupport in a sequencing device that includes reaction chambers such aswells is depicted in FIG. 8. For example, with reference to FIG. 8, anexemplary sequencing device 416 includes an array of wells 418. A beadsupport 406 including a target polynucleotide 402 to be sequenced isattached to a magnetic bead 410 to form a bead assembly 412 that isdesigned for introduction of the bead support with attached targetpolynucleotide into a well. In some embodiments, the magnetic bead 410is attached to the bead support 406 by a double stranded polynucleotidelinkage. In some instances, a linker moiety is hybridized to a portionof the target polynucleotide on the bead support 406. In this example,the linker moiety attaches to a complementary linker moiety on themagnetic bead 410. In another example, the template polynucleotide usedto form the target nucleic acid attached to beads 406 includes a linkermoiety that attaches to the magnetic bead 410. In another example, thetemplate polynucleotide complementary to target polynucleotide attachedto the bead support 406 is generated from a primer that is modified witha linker that attaches to the magnetic bead 410. The linker moietyattached to the polynucleotide and the linker moiety attached to themagnetic bead are, in some embodiments, complementary to and attach toeach other. In an example, the linker moieties have affinity andinclude: an avidin moiety and a biotin moiety; an antigenic epitope andan antibody or immunologically reactive fragment thereof; an antibodyand a hapten; a digoxigen moiety and an anti-digoxigen antibody; afluorescein moiety and an anti-fluorescein antibody; an operator and arepressor; a nuclease and a nucleotide; a lectin and a polysaccharide; asteroid and a steroid-binding protein; an active compound and an activecompound receptor; a hormone and a hormone receptor; an enzyme and asubstrate; an immunoglobulin and protein A; or an oligonucleotide orpolynucleotide and its corresponding complement. In a particularexample, the linker moiety attached to the polynucleotide includesbiotin and the linker moiety attached to the magnetic bead includesstreptavidin.

As illustrated in the embodiment depicted in FIG. 8, a plurality of beadsupports 404 is placed in a solution along with a plurality ofpolynucleotides 402 (target or template polynucleotides). The pluralityof bead supports 404 is activated or otherwise prepared to bind with thepolynucleotides 402. For example, the bead supports 404 include anoligonucleotide (capture primer) complementary to a portion of apolynucleotide of the plurality of polynucleotides 402. In anotherexample, the bead supports 404 are modified with target polynucleotides402 using techniques such as biotin-streptavidin binding. In aparticular embodiment 400, the hydrophilic bead particles andpolynucleotides are subjected to polymerase chain reaction (PCR)amplification or recombinase polymerase amplification (RPA). Thetemplate polynucleotide will hybridize to the capture primer. Thecapture primer is extended to form beads 406 that include a targetpolynucleotide attached thereto. Other beads 408 may remain unattachedto a target nucleic acid and other template polynucleotides may be freefloating in solution. Various methods are available for seeding the beadsupports and capture by the magnetic beads. For example, turning to FIG.9 at 502, a template polynucleotide (B*-A) is captured by a captureprobe (B) attached to a bead support 510. The capture probe (B) isextended complementary to the template polynucleotide producing B-A*.Optionally, the resultant double-stranded polynucleotide is denaturedremoving the template nucleic acid (B*-A) and leaving a single-stranded(B-A*) attached to the bead support 510. As illustrated at 504 of FIG.9, a primer (A) modified with a linker moiety, such as biotin, ishybridized to a portion (A*) of the nucleic acid (B-A*) attached to thebead support 510. Optionally, the primer (A) is extended to form acomplementary nucleic acid (A-B*). As shown at 506, a magnetic bead 512is introduced to the solution. The magnetic bead 512 includes a linkercomplementary to the linker moiety attached to the primer (A). In thisexample, the linker attached to the primer (A) is biotin and themagnetic bead 512 is coated with streptavidin. The magnetic bead 512 maybe utilized to clean the solution and to assist with deposition of thebead support 510 and the attached nucleic acid (B-A*) into a well of asequencing device. As illustrated in 508 of FIG. 9, in some instancesdouble-stranded polynucleotide of 506 is denatured, resulting in thedehybridization of the nucleic acid (B*-A) from the nucleic acid (B-A*)attached to the bead support 510. As such, the bead support 510 isdeposited into the wells of the sequencing device and has a singlestranded target nucleic acid (B-A*). Alternatively, the linker modifiedprobe (A) may not be extended to form a complementary polynucleotidewith a length the polynucleotide (B-A*). Extension reactions can becarried out using polymerase chain reaction (PCR), recombinasepolymerase amplification (RPA), or other amplification reactions.

Turning back to FIG. 8, in an embodiment in which a magnetic bead isutilized to assist with deposition of the bead support and the attachednucleic acid into a well of a sequencing device, bead assemblies 412 areapplied over a substrate 416 of a sequencing device that includes wells418. In an example, a magnetic field can be applied to the substrate 416to draw the magnetic beads 410 of the bead assembly 412 towards thewells 418. The bead support 406 enters the well 418. For example, amagnet can be moved in parallel to a surface of the substrate 416resulting in the deposition of the bead support 406 in the wells 418.The bead assembly 412 is denatured to remove the magnetic bead 410leaving the bead support 406 in the well 418. For example, hybridizeddouble-stranded DNA of the bead assembly 412 can be denatured usingthermal cycling or ionic solutions to release the magnetic bead 410 andtemplate polynucleotides having a linker moiety attached to the magneticbead 410. Optionally, the target polynucleotides 406 can be amplified,such as in a templating reaction, while in the well 418, to provide abead support 414 with multiple copies of the target polynucleotides. Inparticular, the bead 414 has a monoclonal population of targetpolynucleotides. Such amplification reactions are performed, forexample, using polymerase chain reaction (PCR) amplification,recombination polymerase amplification (RPA) or a combination thereof.In a particular embodiment, an enzyme such as a polymerase is present,bound to, or is in close proximity to the particles or beads. In anexample, a polymerase is present in solution or in the well tofacilitate duplication of the polynucleotide. A variety of nucleic acidpolymerases may be used in the methods described herein. In an exemplaryembodiment, the polymerase can include an enzyme, fragment or subunitthereof, which can catalyze duplication of the polynucleotide. Inanother embodiment, the polymerase can be a naturally-occurringpolymerase, recombinant polymerase, mutant polymerase, variantpolymerase, fusion or otherwise engineered polymerase, chemicallymodified polymerase, synthetic molecules, or analog, derivative orfragment thereof. While the polynucleotides of bead support 414 areillustrated as being on a surface, the polynucleotides may extend withinthe bead support 414. Hydrogel and hydrophilic particles having a lowconcentration of polymer relative to water may include polynucleotidesegments on the interior of and throughout the bead support 414 orpolynucleotides may reside in pores and other openings. In particular,in some examples, the bead support 414 permits diffusion of enzymes,nucleotides, primers and reaction products used to monitor the reaction.A high number of polynucleotides per particle generally produces abetter signal.

In some embodiments, sequencing includes extending a template nucleicacid molecule or amplified template nucleic acid molecule, or extendinga sequencing primer hybridized to a template or amplified template, vianucleotide incorporation by a polymerase. In some embodiments,sequencing includes sequencing a template or amplified template that isattached to a support by contacting the template or extended primer witha sequencing primer, a polymerase, and at least one type of nucleotide.In some embodiments, the sequencing includes contacting the template, oramplified template, or extended primer, with a sequencing primer, apolymerase, and with only one type of nucleotide that does not includean extrinsic label or a chain terminating group. In some embodiments, asequencing reaction is conducted using at least one sequencing primerthat hybridizes to any portion of the polynucleotide constructs,including a nucleic acid adapter or a target polynucleotide sequence.

Returning to FIG. 8, in an example, a sequencing primer is added to thewells 418 or the bead support 414 is pre-exposed to the primer prior toplacement in the well 418. In particular, the bead support 414 includesbound sequencing primer. The sequencing primer and polynucleotide form anucleic acid duplex including the polynucleotide (e.g., a templatenucleic acid) hybridized to the sequencing primer. The nucleic acidduplex is an at least partially double-stranded polynucleotide. Enzymesand nucleotides are provided to the well 418 to facilitate detectablereactions, such as nucleotide incorporation. In some embodiments,sequencing involves detecting nucleotide addition. Methods of detectingnucleotide addition include fluorescent emission methods or iondetection methods. For example, a set of fluorescently labelednucleotides is provided to the system 416 and migrates to the well 418.Excitation energy is also provided to the well 418. When a nucleotide iscaptured by a polymerase and added to the end of an extending primer, alabel of the nucleotide fluoresces, indicating which type of nucleotideis added. In an alternative example, solutions including a single typeof nucleotide are fed sequentially. In response to nucleotide addition,the pH within the local environment of the well 418 may change. Such achange in pH is detectable by ion sensitive field effect transistors(ISFET). As such, a change in pH may be used to generate a signalindicating the order of nucleotides complementary to the polynucleotideof the particle 410.

In a typical embodiment of ion-based nucleic acid sequencing, nucleotideincorporations is detected by detecting the presence and/orconcentration of hydrogen ions generated by polymerase-catalyzedextension reactions. In one embodiment, template nucleic acid molecules,optionally pre-bound to a sequencing primer and/or a polymerase, can beloaded into reaction chambers after which repeated cycles of nucleotideaddition and washing are carried out. In some embodiments, suchtemplates are attached as substantially monoclonal populations to asupport, such as particles, bead, or the like, and said substantiallymonoclonal populations are loaded into reaction chambers.

In each addition step of the cycle, the polymerase extends the primer byincorporating added nucleotide only if the next base in the template isthe complement of the added nucleotide in solution. If there is onecomplementary base on the template nucleic acid molecule, there is oneincorporation, if there two complementary bases in a row on the templatenucleic acid molecule, there are two incorporations, if three, there arethree incorporations, and so on. With each such incorporation, there isa hydrogen ion released, and collectively a population of templatesreleasing hydrogen ions changes the local pH of the reaction chamber.The production of hydrogen ions is monotonically related to the numberof contiguous complementary bases in the template (as well as the totalnumber of template molecules with primer and polymerase that participatein an extension reaction). Thus, when there are a number of contiguousidentical complementary bases in the template (i.e. a homopolymerregion), the number of hydrogen ions generated, and therefore themagnitude of the local pH change, is proportional to the number ofcontiguous identical complementary bases. If the next base in thetemplate is not complementary to the added nucleotide, then noincorporation occurs and no hydrogen ion is released. In someembodiments, after each step of adding a nucleotide, an additional stepis performed, in which an unbuffered wash solution at a predetermined pHis used to remove the nucleotide of the previous step in order toprevent misincorporations in later cycles. In some embodiments, aftereach step of adding a nucleotide, an additional step is performedwherein the reaction chambers are treated with a nucleotide-destroyingagent, such as apyrase, to eliminate any residual nucleotides remainingin the chamber, which may result in spurious extensions in subsequentcycles.

In particular embodiments, a sequencing system includes a well, or aplurality of wells, disposed over a sensor pad of an ionic sensor, suchas a field effect transistor (FET). In some embodiments, the FET is aFET array. The FET or array may include, for example, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷ or more FETs. In embodiments, a system includes one ormore polymeric particles loaded into a well which is disposed over asensor pad of an ionic sensor (e.g., FET), or one or more polymericparticles loaded into a plurality of wells which are disposed oversensor pads of ionic sensors (e.g., FET). In embodiments, an FET is achemFET or an ISFET. A “chemFET” or chemical field-effect transistor,includes a type of field effect transistor that acts as a chemicalsensor. The chemFET has the structural analog of a MOSFET transistor,where the charge on the gate electrode is applied by a chemical process.An “ISFET” or ion-sensitive field-effect transistor, is used formeasuring ion concentrations in solution; when the ion concentration(such as H+) changes, the current through the transistor changesaccordingly. In some embodiments, one or more microfluidic structuresare fabricated above the FET sensor array to provide for containment orconfinement of a biological or chemical reaction. For example, in oneimplementation, the microfluidic structure(s) are configured as one ormore wells (or wells, or reaction chambers, or reaction wells, as theterms are used interchangeably herein) disposed above one or moresensors of the array, such that the one or more sensors over which agiven well is disposed detect and measure analyte presence, level, orconcentration in the given well. In embodiments, there can be a 1:1correspondence of FET sensors and reaction wells.

Returning to FIG. 8, in another example, a well 418 of the array ofwells is operatively connected to measuring devices. For example, forfluorescent emission methods, a well 418 may be operatively coupled to alight detection device. In the case of ionic detection, the lowersurface of the well 418 may be disposed over a sensor pad of an ionicsensor, such as a field effect transistor.

In some embodiments, different kinds of nucleotides are addedsequentially to the reaction chambers, so that each reaction is exposedto the different nucleotides one at a time. For example, nucleotides areadded in the following sequence: dATP, dCTP, dGTP, dTTP, dATP, dCTP,dGTP, dTTP, and so on; with each exposure followed by a wash step. Thecycles may be repeated for 50 times, 100 times, 200 times, 300 times,400 times, 500 times, 750 times, or more, depending on the length ofsequence information desired.

In some embodiments, the methods (and related compositions, systems, andkits) include detecting the presence of one or more nucleotideincorporation byproducts at a site of the array, optionally using theFET. In some embodiments, the methods include detecting a pH changeoccurring within the at least one reaction chamber, optionally using theFET. In some embodiments, the disclosed methods further includedetecting a change in ion concentration in at least one of the sites asa result of the at least one amplification cycle.

An exemplary system involving sequencing via detection of ionicbyproducts of nucleotide incorporation is the Ion Torrent PGM™, Proton™or S5™ sequencer (Thermo Fisher Scientific), which is an ion-basedsequencing system that sequences nucleic acid templates by detectinghydrogen ions produced as a byproduct of nucleotide incorporation.Typically, hydrogen ions are released as byproducts of nucleotideincorporations occurring during template-dependent nucleic acidsynthesis by a polymerase. The Ion Torrent PGM™, Proton™, or S5™sequencer detects the nucleotide incorporations by detecting thehydrogen ion byproducts of the nucleotide incorporations. In someembodiments, the Ion Torrent PGM™, Proton™ or S5™ sequencer includes aplurality of template polynucleotides to be sequenced, each templatedisposed within a respective sequencing reaction well in an array. Thewells of the array are each be coupled to at least one ion sensor thatdetects the release of H+ ions or changes in solution pH produced as abyproduct of nucleotide incorporation. The ion sensor comprises a fieldeffect transistor (FET) coupled to an ion-sensitive detection layer thatsenses the presence of H+ ions or changes in solution pH. The ion sensorprovides output signals indicative of nucleotide incorporation which maybe represented as voltage changes whose magnitude correlates with the H+ion concentration in a respective well or reaction chamber. Differentnucleotide types are flowed serially into the reaction chamber, and maybe incorporated by the polymerase into an extending primer (orpolymerization site) in an order determined by the sequence of thetemplate. For example, a sequencing system containing a fluidics circuitis connected by inlets to at least two reagent reservoirs, a wastereservoir, and to a biosensor by a fluid pathway for fluidiccommunication. Reagents from reservoirs are driven to the fluidiccircuit by a variety of methods including pressure, pumps, such assyringe pumps, gravity feed, and the like, and are selected by controlof valves. Reagents from the fluidics circuit are driven through thevalves receiving signals from a control system. The control systemincludes controllers for valves, which generate signals for opening andclosing via an electrical connection. The control system also includescontrollers for other components of the system, such as a wash solutionvalve connected thereto by an electrical connection and a referenceelectrode. Each nucleotide incorporation is accompanied by the releaseof H+ ions in the reaction well, along with a concomitant change in thelocalized pH. The release of H+ ions is registered by the FET of thesensor, which produces signals indicating the occurrence of thenucleotide incorporation. Nucleotides that are not incorporated during aparticular nucleotide flow may not produce signals. The amplitude of thesignals from the FET may also be correlated with the number ofnucleotides of a particular type incorporated into the extending nucleicacid molecule thereby permitting homopolymer regions to be resolved.Thus, during a run of the sequencer multiple nucleotide flows into thereaction chamber along with incorporation monitoring across amultiplicity of wells or reaction chambers permits the instrument toresolve the sequence of many nucleic acid templates simultaneously.

In some embodiments, methods of downstream analysis include sequencingat least some of the population of amplicons in parallel. Optionally,the multiple templates/amplified templates/extended first primerssituated at different sites of the array are sequenced in parallel.

In some embodiments, the sequencing includes binding a sequencing primerto the nucleic acid molecules of at least two different template nucleicacid molecules, or at least two different substantially monoclonalpopulations. In some embodiments, the sequencing includes incorporatinga nucleotide onto the 3′ OH of the sequencing primer using thepolymerase. Optionally, the incorporating includes forming at least onenucleotide incorporation byproduct.

In the embodiments of the present disclosure involving distribution oftemplate nucleic acid molecules into the wells of an isFET array andsubsequent amplification of templates inside the wells of the array, anoptional step of downstream analysis is performed after theamplification that quantifies the number of sites or wells that includeamplification product. In some embodiments, the products of the nucleicacid amplification reactions are detected in order to count the numberof sites or wells that include an amplified template. In someembodiments, after the amplification, at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the wellsincludes a substantially monoclonal population of template nucleic acidmolecules.

In some embodiments, the templated supports are distributed intodistributed into separate reaction chambers (e.g., an array of wells)for sequencing. In some embodiments, at least 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the reactionchambers have a templated support with substantially monoclonalpopulations of template nucleic acid molecules. In some embodiments,between about 25% and 95% of the reaction chambers have a templatedsupport with substantially monoclonal populations of template nucleicacid molecules, for example between about 25% and 85%, between about 25%and 75%, between about 35% and 95%, between about 35% and 85%, betweenabout 35% and 75%, between about 45% and 95%, between about 45% and 85%,between about 45% and 75%, between about 55% and 95%, between about 55%and 85%, between about 55% and 75%, between about 65% and 95%, betweenabout 65% and 85%, between about 65% and 75%, between about 75% and 95%,between about 75% and 85%, or between about 85% and 95% of the reactionchambers have a templated support with substantially monoclonalpopulations of template nucleic acid molecules. In some embodiments, theseparate reaction chambers are wells on a sequencing chip. In someembodiments, no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% or 25% ofthe wells give low quality results, where low quality wells aredetermined by the sequencing method.

In various embodiments, the methods, systems, and computer readablemedia described herein may advantageously be used to process and/oranalyze data and signals obtained from electronic or charged-basednucleic acid sequencing. In electronic or charged-based sequencing (suchas, pH-based sequencing), a nucleotide incorporation event may bedetermined by detecting ions (e.g., hydrogen ions) that are generated asnatural by-products of polymerase-catalyzed nucleotide extensionreactions. This may be used to sequence a template nucleic acidmolecule, which may be a fragment of a nucleic acid sequence ofinterest, for example, and which may be directly or indirectly attachedas a substantially monoclonal population of nucleic acid molecules on asupport, such as a particle, microparticle, bead, etc. The sample ortemplate nucleic acid may be operably associated to a primer andpolymerase and may be subjected to repeated cycles or “flows” ofnucleotide addition (which may be referred to herein as “nucleotideflows” from which nucleotide incorporations may result) and washing. Theprimer may be annealed to the sample or template so that the 3′ end ofthe primer is extended by a polymerase whenever nucleotidescomplementary to the next base in the template are added. Then, based onthe known sequence of nucleotide flows and on measured output signals ofthe chemical sensors indicative of ion concentration during eachnucleotide flow, the identity of the type, sequence and number ofnucleotide(s) associated with a sample nucleic acid present in areaction region coupled to a chemical sensor can be determined.

In the templating reaction, a sufficient number of substantiallymonoclonal or monoclonal populations can be produced to generate atleast 100 MB, 200 MB, 300 MB, 400 MB, 500 MB, 750 MB, 1 GB or 2 GB ofAQ20 sequencing reads on an Ion Torrent PGM™ 314, 316 or 318 sequencer.With respect to related high-throughput systems, a sufficient number ofsubstantially monoclonal or monoclonal amplicons can be produced in asingle amplification reaction to generate at least 100 MB, 200 MB, 300MB, 400 MB, 500 MB, 750 MB, 1 GB, 2 GB, 5 GB, 10 GB or 15 GB of AQ20sequencing reads on an Ion Torrent Proton, S5 or S5XL sequencer. Theterm “AQ20” and its variants, as used herein, refers to a particularmethod of measuring sequencing accuracy in the Ion Torrent PGM™sequencer. Accuracy can be measured in terms of the Phred-like Q score,which measures accuracy on logarithmic scale that: Q10=90%, Q20=99%,Q30=99.9%, Q40=99.99%, and Q50=99.999%. For example, in a particularsequencing reaction, accuracy metrics can be calculated either throughprediction algorithms or through actual alignment to a known referencegenome. Predicted quality scores (“Q scores”) can be derived fromalgorithms that look at the inherent properties of the input signal andmake fairly accurate estimates regarding if a given single base includedin the sequencing “read” will align. In some embodiments, such predictedquality scores are useful to filter and remove lower quality reads priorto downstream alignment. In some embodiments, the accuracy is reportedin terms of a Phred-like Q score that measures accuracy on logarithmicscale such that: Q10=90%, Q17=98%, Q20=99%, Q30=99.9%, Q40=99.99%, andQ50=99.999%. In some embodiments, the data obtained from a givenpolymerase reaction is filtered to measure only polymerase readsmeasuring “N” nucleotides or longer and having a Q score that passes acertain threshold, e.g., Q10, Q17, Q100 (referred to herein as the“NQ17” score). For example, the 100Q20 score can indicate the number ofreads obtained from a given reaction that are at least 100 nucleotidesin length and have Q scores of Q20 (99%) or greater. Similarly, the200Q20 score can indicate the number of reads that are at least 200nucleotides in length and have Q scores of Q20 (99%) or greater.

In some embodiments, the accuracy is calculated based on properalignment using a reference genomic sequence, referred to herein as the“raw” accuracy. This is single pass accuracy, involving measurement ofthe “true” per base error associated with a single read, as opposed toconsensus accuracy, which measures the error rate from the consensussequence which is the result of multiple reads. Raw accuracymeasurements can be reported in terms of “AQ” scores (for alignedquality). In some embodiments, the data obtained from a given polymerasereaction is filtered to measure only polymerase reads measuring “N”nucleotides or longer having a AQ score that passes a certain threshold,e.g., AQ10, AQ17, AQ100 (referred to herein as the “NAQ17” score). Forexample, the 100AQ20 score can indicate the number of reads obtainedfrom a given polymerase reaction that are at least 100 nucleotides inlength and have AQ scores of AQ20 (99%) or greater. Similarly, the200AQ20 score can indicate the number of reads that are at least 200nucleotides in length and have AQ scores of AQ20 (99%) or greater.

In some embodiments, the present disclosure provides kits for nucleicacid amplification in a pre-seeding reaction followed by a templatingreaction. The compositions discussed herein are also amendable to kitformat wherein the primers, and amplification components may be in thesame container, separate containers and in liquid or dehydrated form.The kit can include instructions for performing the methods foramplification of template nucleic acid molecules including a pre-seedingreaction for downstream sequencing methods. In one embodiment, the kitprovides instructions for nucleic acid sequencing preparation.

In some embodiments, a kit that includes at least two containers, atleast one of which includes a primer and at least one of which includesa recombinase. The recombinase and the primer can be in the same ordifferent tubes. In some embodiments, at least one primer is attached toone or more supports. The kits can further include one or morepre-seeded solid supports.

In some embodiments, the container including the recombinase furtherinclude one or more amplification reagents including a recombinaseaccessory protein, a polymerase, dNTPs, and a buffer. In certainembodiments, the kit includes one or more containers with uvsXrecombinase, uvsY recombinase loading protein, gp32 protein, Sau DNApolymerase, dNTPs, ATP, phosphocreatine, and creatine kinase.

In some embodiments, the kits include any combination of: one or moresolid supports, optionally with a population of at least one primerattached, polynucleotides, recombinase, recombinase loading protein,single-stranded binding protein (SSB), polymerase, nucleotides, ATP,phosphocreatine, creatine kinase, hybridization solutions, washingsolutions, buffers and/or cations (e.g., divalent cations). A kit caninclude all or some of these components, typically in at least twoseparate vessels. The kit can include any of the components ofpre-seeding reaction mixtures or templating reaction mixtures.

In certain embodiments, provided is a method for generating a nucleicacid template comprising a specific nucleotide sequence, comprisingobtaining a population of nucleic acid molecules in which each moleculecomprises a first sequence of contiguous nucleotides at the 5′ end ofthe molecule, a second sequence of contiguous nucleotides at the 3′ endof the molecule and a third nucleotide sequence positioned between thefirst and second nucleotide sequences, wherein the first nucleotidesequence and second nucleotide sequence are different from each other,and wherein the first nucleotide sequences of the nucleic acid moleculesare substantially identical and the second nucleotide sequences of thenucleic acid molecules are substantially identical among the population;subjecting the population of nucleic acid molecules to a cycle ofnucleic acid amplification in the presence of a forward primer and areverse primer, wherein the forward primer comprises an oligonucleotidesequence substantially identical to the first nucleotide sequence andthe reverse primer comprises an oligonucleotide sequence complementaryto a subsequence of the 5′ end of the second nucleotide sequence that islinked at the 3′ end of the subsequence to a fourth nucleotide sequencethat is not complementary to the second nucleotide sequence; andsubjecting the products of the cycle of amplification of (b) to a cycleof amplification in the presence of the forward and reverse primers togenerate multiple different nucleic acid products; wherein the reverseprimer includes a nucleotide sequence complementary to the 5′ end of thesecond nucleotide sequence but does not contain a nucleotide sequencecomplementary to the 3′ end of the second nucleotide sequence, and onlyone of the products comprises a sequence of nucleotides complementary tothe fourth nucleotide sequence. In some embodiments the forward primercomprises a modified nucleotide containing an attachment thereto. Insome embodiments the attachment to the modified nucleotide comprisesbiotin. In some embodiments the method further comprises combining thesingle-stranded nucleic acids of the products to a single-strandedoligonucleotide that is substantially identical to the fourth nucleotidesequence under annealing conditions thereby hybridizing the product thatcomprises a sequence of nucleotides complementary to the fourthnucleotide sequence to the single-stranded oligonucleotide that issubstantially identical to the fourth nucleotide sequence to generate apartially double-stranded oligonucleotide-bound product. In someembodiments the single-stranded oligonucleotide that is substantiallyidentical to the fourth nucleotide sequence is attached to a support. Insome embodiments the support is a solid support. In particularembodiments the support is a particle or bead. In some embodiments themethod further comprises extending the 3′ end of the oligonucleotidethat is hybridized to the product that comprises a sequence ofnucleotides complementary to the fourth nucleotide sequence therebygenerating a double-stranded nucleic acid by synthesizing a nucleic acidstrand comprising the single-stranded oligonucleotide that issubstantially identical to the fourth nucleotide sequence and having anucleotide sequence that is complementary to the product to which it ishybridized. In some embodiments the method further comprises separatingthe strands of the double-stranded nucleic acid. In some embodiments thenucleic acid strand comprising the single-stranded oligonucleotide thatis substantially identical to the fourth nucleotide sequence is attachedto a support at the 5′ end of the strand through the portion of thestrand that is the oligonucleotide sequence. In some embodiments themethod further comprises isolating the nucleic acid strand attached tothe support by removing the support from any other nucleic acids thatare not bound to the support.

In certain aspects, provided is a method for generating nucleic acidmolecules comprising a specific nucleotide sequence, comprisingobtaining a population of nucleic acid molecules in which each moleculecomprises a first sequence of contiguous nucleotides at the 5′ end ofthe molecule, a second sequence of contiguous nucleotides at the 3′ endof the molecule and a third nucleotide sequence positioned between thefirst and second nucleotide sequences, wherein the first nucleotidesequence and second nucleotide sequence are different from each other,and wherein the first nucleotide sequences of the nucleic acid moleculesare substantially identical and the second nucleotide sequences of thenucleic acid molecules are substantially identical among the population,and subjecting the population of nucleic acid molecules to two or morecycles of nucleic acid amplification in the presence of one or moreforward primers comprising an oligonucleotide sequence substantiallyidentical to the first nucleotide sequence and a reverse primer that isblocked at the 3′ end and comprises an oligonucleotide sequencecomplementary to the second nucleotide sequence that is linked at the 5′end of the oligonucleotide sequence to a fourth nucleotide sequence thatis not complementary to the second nucleotide sequence to generatenucleic acid products in which substantially all of the productscomprise a sequence of nucleotides complementary to the fourthnucleotide sequence. In some embodiments the forward primer comprises amodified nucleotide containing an attachment thereto. In someembodiments the attachment to the modified nucleotide comprises biotin.In some embodiments the method further comprises exposingsingle-stranded nucleic acids of the products of (b) to asingle-stranded oligonucleotide that is substantially identical to thefourth nucleotide sequence under annealing conditions therebyhybridizing the products of (b) that comprise a sequence of nucleotidescomplementary to the fourth nucleotide sequence to the single-strandedoligonucleotide that is substantially identical to the fourth nucleotidesequence to generate a partially double-stranded oligonucleotide-boundproduct. In some embodiments the single-stranded oligonucleotide that issubstantially identical to the fourth nucleotide sequence is attached toa support. In some embodiments the support is a solid support. Incertain embodiments the support is a particle or bead. In someembodiments the method further comprises extending the 3′ end of theoligonucleotide that is hybridized to the product that comprises asequence of nucleotides complementary to the fourth nucleotide sequencethereby generating a double-stranded nucleic acid by synthesizing anucleic acid strand comprising the single-stranded oligonucleotide thatis substantially identical to the fourth nucleotide sequence and havinga nucleotide sequence that is complementary to the product to which itis hybridized. In some embodiments the method further comprisesseparating the strands of the double-stranded nucleic acid. In someembodiments the nucleic acid strand comprising the single-strandedoligonucleotide that is substantially identical to the fourth nucleotidesequence is attached to a support at the 5′ end of the strand throughthe portion of the strand that is the oligonucleotide sequence. In someembodiments the method further comprises isolating the nucleic acidstrand attached to the support by removing the support from any othernucleic acids that are not bound to the support.

In one aspect, provided is a templating reaction mixture comprising apopulation of pre-seeded solid supports, nucleotides, a recombinase, anda polymerase, wherein the population of pre-seeded solid supportscomprise between 10 and 50,000 substantially monoclonal template nucleicacid molecules comprising a first primer attached thereto and furthercomprise attached first primers not bound to template nucleic acidmolecules, wherein the reaction mixture does not comprise a cationcapable of initiating a recombinase-polymerase amplification reaction,and wherein at least 95% of the template nucleic acid molecules in thereaction mixture are attached to the one or more pre-seeded solidsupports. In some embodiments the template nucleic acid moleculescomprise two or more template nucleic acid molecules with differentsequences. In some embodiments the substantially monoclonal templatenucleic acid molecules comprise a proximal segment having 20 to 50identical nucleotides attaching a template nucleic acid molecule to asolid support. In some embodiments the substantially monoclonal templatenucleic acid molecules comprise fewer than 100 identical nucleotides ata proximal segment attaching a template nucleic acid molecule to a solidsupport. In some embodiments the pre-seeded solid supports arepre-seeded beads. In some embodiments each pre-seeded solid support ofthe population of pre-seeded solid supports has between 10 and 10,000substantially monoclonal template nucleic acid molecules attachedthereto and wherein the one or more pre-seeded solid supports are beads.In some embodiments less than 10% of the substantially monoclonaltemplate nucleic acid molecules on each pre-seeded solid supportcomprise 50-100 identical nucleotides attaching them to the pre-seededsolid support. In some embodiments the templating reaction mixturefurther comprises a recombinase-accessory protein. In some embodimentsthe recombinase-accessory protein is a single-stranded binding proteinand/or a recombinase-loading protein. In some embodiments the pre-seededsolid supports are generated using a first recombinase-polymeraseamplification (RPA) reaction. In some embodiments the first RPA reactionis performed by incubating an RPA reaction mixture for 2 to 5 minutes ata temperature between 35° C. and 45° C. In some embodiments thepre-seeding reaction mixture further comprises a population of identicalsecond primers in solution, and wherein the template nucleic acidmolecules comprise a primer binding site for the first primer at or neara first terminus. In some embodiments the reaction mixture furthercomprises a cation capable of initiating the recombinase-polymeraseamplification reaction.

In another aspect of the invention, provided is a method of generatingone or more template nucleic acid populations on a solid support,comprising: a) obtaining a population of nucleic acid molecules whereineach molecule comprises a first adapter sequence of contiguousnucleotides at the 5′ end of the molecule, a second adapter sequence ofcontiguous nucleotides at the 3′ end of the molecule and a thirdnucleotide sequence positioned between the first and second nucleotidesequences, wherein the first adapter nucleotide sequence and the secondadapter nucleotide sequence are different; wherein the first adapternucleotide sequences of the nucleic acid molecules are substantiallyidentical, the second adapter nucleotide sequences of the nucleic acidmolecules are substantially identical, and the nucleotide sequencepositioned between the first and second nucleotide sequences differamong the population; and wherein the first adapter nucleotide sequencecomprises a first linker moiety attached thereto; b) generating singlestrands of the population of nucleic acid molecules and contacting thesingle-stranded nucleic acids with solid supports under annealingconditions to generate solid supports having single-stranded nucleicacids attached thereto through hybridization with the second adaptersequence of the nucleic acids, wherein the solid supports comprise aplurality of primer oligonucleotides having a nucleotide sequencecomplementary to the second adapter sequence of the nucleic acidsimmobilized thereto; c) extending the immobilized primers of the solidsupport that are hybridized to a nucleic acid strand to generatedouble-stranded nucleic acids bound to the solid supports; d) subjectingthe nucleic acid molecules bound to the solid supports to a cycle ofnucleic acid amplification in the presence of a first primer comprisingan oligonucleotide sequence substantially identical to the first adapternucleotide sequence and a linker moiety attached thereto, wherein alimited number of single-stranded nucleic acids attach to each solidsupport, and the nucleic acids attached to an individual support aresubstantially monoclonal; e) combining the solid supports having nucleicacids bound thereto with magnetic beads comprising a second linkingmoiety to which the first linking moiety attaches to generate solidsupport-magnetic bead assemblies; f) applying a magnetic field to thebead assemblies thereby separating the bead assemblies from elementsthat do not include a magnetic bead; g) releasing the solid supportshaving nucleic acids attached thereto from the magnetic beads; h)combining the solid supports having nucleic acids attached thereto withmagnetic beads wherein the nucleic acids on the solid supports will notattach to the magnetic beads; i) delivering the combined solid supportshaving nucleic acids attached thereto and magnetic beads to a surfacecomprising microwells and applying a magnetic field to the surfacewhereby the solid supports having nucleic acids attached thereto areloaded into separate microwells; and j) subjecting the nucleic acidmolecules bound to the solid supports in the microwells to at least onecycle of nucleic acid amplification in the presence of a first primer insolution, wherein the first primer comprises an oligonucleotide sequencesubstantially identical to the first adapter nucleotide sequence andwherein the amplification generates at least 10-fold more nucleic acidsattached to the solid support than were attached after the amplificationset out in (d); wherein the reverse primer includes a nucleotidesequence complementary to the 5′ end of the second nucleotide sequencebut does not contain a nucleotide sequence complementary to the 3′ endof the second nucleotide sequence. In some embodiments the number ofsolid supports in (b) exceeds the number of nucleic acid molecules by afactor of at least 2. In some embodiments the number of solid supportsin (b) exceeds the number of nucleic acid molecules by a factor of atleast 5. In some embodiments the amplification in (j) generates at least100,000-fold more nucleic acids attached to the solid support than wereattached after the amplification in (d). In some embodiments theamplification in (j) generates at least 1,000,000-fold more nucleicacids attached to the solid support than were attached after theamplification in (d). In some embodiments the method further comprisessubjecting the amplified nucleic acids attached to the solid supports tonucleic acid sequencing. In some embodiments the sequencing processproduces at least 60 million sequence reads which are at least 300nucleotides in length. In some embodiments the sequencing processproduces at least 80 million sequence reads which are at least 100nucleotides in length. In some embodiments the sequencing processproduces at least 80 million sequence reads between about 100 and about400 nucleotides in length.

In another aspect, provided is a method of generating one or moretemplate nucleic acid populations on a solid support, comprising: a)obtaining a population of nucleic acid molecules in which each moleculecomprises a first adapter sequence of contiguous nucleotides at the 5′end of the molecule, a second adapter sequence of contiguous nucleotidesat the 3′ end of the molecule and a third nucleotide sequence positionedbetween the first and second nucleotide sequences, wherein the firstadapter nucleotide sequence and the second adapter nucleotide sequenceare different and the first adapter nucleotide sequences of the nucleicacid molecules are substantially identical and the second adapternucleotide sequences of the nucleic acid molecules are substantiallyidentical and wherein the first adapter nucleotide sequence is modifiedto comprise a first linker moiety attached thereto; b) generating singlestrands of the population of nucleic acid molecules and contacting thesingle-stranded nucleic acids with solid supports under annealingconditions to generate solid supports having single-stranded nucleicacids attached thereto through hybridization with the second adaptersequence of the nucleic acids, wherein the solid supports comprise aplurality of primer oligonucleotides having a nucleotide sequencecomplementary to the second adapter sequence of the nucleic acidsimmobilized thereto and the number of solid supports exceeds the numberof nucleic acid molecules by a factor of at least 5. c) transferring thesolid supports having nucleic acids attached thereto to a surfacecomprising microwells whereby the solid supports having nucleic acidsattached thereto are individually loaded into separate microwells; d)extending the immobilized primers of the solid support that arehybridized to a nucleic acid strand to generate double-stranded nucleicacids bound to the solid supports; e) subjecting the nucleic acidmolecules bound to the solid supports to a cycle of nucleic acidamplification in the presence of a first primer in solution, wherein (1)the first primer comprises an oligonucleotide sequence substantiallyidentical to the first adapter nucleotide sequence and is modified tocomprise a linker moiety attached thereto, (2) the amplificationgenerates a limited number of additional single-stranded nucleic acidsattached to the solid supports through hybridization between the primeroligonucleotides having a nucleotide sequence complementary to thesecond adapter sequence immobilized on the solid supports and the secondadapter sequence of the nucleic acids, (3) the nucleic acids attached toan individual support are substantially monoclonal, and (4) theamplification is conducted in the presence of a composition thatattaches to the linker moiety of the first adapter; f) stopping theamplification of (e) and removing the composition that attaches to thelinker moiety of the first adapter from the microwells; and g)subjecting the nucleic acid molecules bound to the solid supports in themicrowells to at least one cycle of nucleic acid amplification in thepresence of a first primer in solution, wherein the first primercomprises an oligonucleotide sequence substantially identical to thefirst adapter nucleotide sequence and wherein the amplificationgenerates at least 1000-fold more nucleic acids attached to the solidsupport than were attached after the amplification of step (e); whereinthe reverse primer includes a nucleotide sequence complementary to the5′ end of the second nucleotide sequence but does not contain anucleotide sequence complementary to the 3′ end of the second nucleotidesequence and the nucleic acids attached to an individual support aresubstantially monoclonal.

In another aspect, provided is a method of preparing a device fornucleic acid analysis, the method comprising: generating a templatenucleic acid including a capture sequence portion, a template portion,and primer portion modified with a linker moiety; capturing the templatenucleic acid on a bead support having a plurality of capture primerscomplementary to the capture sequence portion of the template nucleicacid, the capture primers hybridizing to the capture sequence portion ofthe template nucleic acid; linking the captured template nucleic acid toa magnetic bead having second linker moiety to form a bead assembly, thesecond linker moiety attaching to the first linker moiety; and loadingthe bead assembly into a well of the sequencing device using a magneticfield. In some embodiments the method further comprises extending thecapture primer complementary to the template nucleic acid to form asequence target nucleic acid attached to the bead support. In someembodiments the method further comprises denaturing the template nucleicacid and the sequence target nucleic acid to release the magnetic beadfrom the bead support. In certain embodiments denaturing includesenzymatic denaturing. In certain embodiments denaturing includesdenaturing in the presence of an ionic solution. In some embodiments themethod further comprises washing the magnetic bead from the sequencingdevice. In some embodiments the method further comprises amplifying thesequence target nucleic acid to form a population of sequence targetnucleic acids on the bead support in the well. In some embodimentsamplifying include performing recombinase polymerase amplification(RPA). In some embodiments performing RPA includes performing RPA for afirst period, washing, and performing RPA for a second period, the firstperiod shorter than the second period. In some embodiments generatingincludes extending a linker modified primer complementary to a targetnucleic acid. In certain embodiments generating comprises amplifying atarget nucleic acid having a first primer portion, a target portion, anda second primer portion in the presence of a bead support having acapture primer, a linker modified first primer complementary to thefirst primer portion, and a second primer having a portion complementaryto at least a portion of the second primer portion, the second primerhaving a capture primer portion ligated to the portion and complementaryto the capture primer, wherein the bead support capture primer isextended to include a sequence of the target nucleic acid. In someembodiments amplifying includes performing three polymerase chainreaction (PCR) cycles.

In another aspect, provided is a method of preparing a sequencingdevice, the method comprising: generating a template nucleic acidincluding a capture sequence portion, a template portion, and primerportion; capturing the template nucleic acid on a bead support coupledto a capture primer complementary to the capture sequence portion, thecapture primer hybridizing to the capture sequence portion of thetemplate nucleic acid; extending the capture primer complementary to thetemplate nucleic acid to form a target nucleic acid complementary to andhybridized to the template nucleic acid; denaturing to separate thehybridized template nucleic acid and the target nucleic acid;hybridizing a linker modified primer to the target nucleic acid on thebead support, the linker modified primer including a linker moiety;extending the linker modified primer complementary to the target nucleicacid; coupling a magnetic bead to the linker moiety, the magnetic beadhaving a second linker moiety attaching to the linker moiety; anddepositing the bead support into a well of a sequencing device using amagnetic field. In some embodiments the method further comprisesdenaturing the template nucleic acid and the sequence target nucleicacid to release the magnetic bead from the bead support. In someembodiments denaturing includes enzymatic denaturing. In someembodiments denaturing includes denaturing in the presence of an ionicsolution. In some embodiments the method further comprises washing themagnetic bead from the sequencing device. In some embodiments the methodfurther comprises amplifying the sequence target nucleic acid to form apopulation of sequence target nucleic acids on the bead support in thewell. In some embodiments amplifying include performing recombinasepolymerase amplification (RPA). In some embodiments performing RPAincludes performing RPA for a first period, washing, and performing RPAfor a second period, the first period shorter than the second period. Insome embodiments generating includes extending a linker modified primercomplementary to a target nucleic acid. In some embodiments theextending includes performing polymerase chain reaction (PCR).

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how touse the embodiments of the present teachings, and are not intended tolimit the scope of the disclosure nor are they intended to representthat the Examples below are all of the experiments or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by volume, and temperature is indegrees Centigrade. It should be understood that variations in themethods as described can be made without changing the fundamentalaspects that the Examples are meant to illustrate.

EXAMPLES Example 1 Pre-Seeding P1 Ion Sphere Particles (ISPs) with aSingle-Cycle PCR Before Bulk Isothermal Amplification

This example provides a method to pre-seed ISPs with monoclonaltemplates before isothermal amplification for downstream next-generationsequencing. These pre-seeded ISPs require no additional templates insolution during the isothermal amplification step and are able togenerate templates that produce better sequencing results than ISPs withno pre-seeded template (non-template control or “NTC”). Ion Torrent ISPswith forward P1 adapters (“P1 ISPs”) were pre-seeded with different copynumbers of monoclonal templates and amplified in bulk isothermalamplification (“Bulk IA”). The pre-seeded ISPs were compared to notemplate control (“NTC”) ISPs, which had the same templatepolynucleotides added during the templating reaction instead of duringthe pre-seeding reaction before the templating reaction. Sequencingresults from the different amplification reactions were compared usingvarious metrics.

Generating Template Nucleic Acid Molecules

DNA was amplified from genomic DNA using PCR primers 1, 12, and 13 fromthe Oncomine Focus Assay (OFA) (Thermo Fisher Scientific, Waltham,Mass.). Adapters that facilitated binding to immobilized primer B and aprimer A in solution used in the seeding reaction, were added to theamplicons during 10 cycles of a second tailed PCR. The amplificationgenerated 3 templates: OFA 1 AB, 19.2 ng/μl (148 nM); OFA 12 AB, 15.6ng/μl (100 nM); and OFA 13 AB, 17.2 ng/μl (76 nM).

Pre-Seeding Reaction

Dilutions of the OFA 1 AB, OFA 12 AB, and OFA 13 AB templates were madeand used to generate separate pre-seeded P1 ISPs in a pre-seedingreaction. The pre-seeding reaction included 6.67 μl P1 ISPs withimmobilized primer B (400,000,000 ISPs), 88.33 μl 1× Platinum HiFi Mix(Thermo Fisher Scientific, Waltham, Mass.), and 5 μl of the appropriatetemplate dilution to generate a desired number of substantiallymonoclonal template molecules (copy numbers 70, 665, and 4,170 for P1ISPs). The ISP pre-seeding reaction mixtures were placed in athermocycler for a denaturation step of 98° C. for 2 min followed by 98°C. for 25 sec and 56° C. for 10 min to generate pre-seeded ISPs. Tocheck the relative sizes of the ISPs, 1 μl of each of the pre-seeded P1ISPs was diluted in 999 μl Annealing Buffer (Ion PGM™ Hi-Q™ ViewSequencing Solutions, (Part No. A30275)) and analyzed using a GuavaeasyCyte Flow Cytometer (EMD Millipore, Billerica, Mass.). The NTC P1ISPs were also analyzed for relative size in the absence of boundtemplate.

The remaining pre-seeded P1 ISPs were washed twice in Ion OneTouch WashSolution wash buffer in a total volume of 1 ml. After the first wash,the samples were centrifuged at >21,000 g for 8 min and the supernatantwas removed to leave ˜100 μl. After the second wash and centrifugation,the supernatant was removed to leave ˜50 μl sample in the tube. Thesample was then treated with 300 μl of freshly prepared melt offsolution (125 mM NaOH, 0.1% Tween 20) and thoroughly vortexed beforeincubation for 5 minutes at room temperature. These samples were thenwashed three times with nuclease-free (NF) H₂O in a final volume of 1ml. After each wash, the samples were centrifuged at >21,000 g for 8 mMand the supernatants were removed to leave ˜100 μl. To check the size ofthe pre-seeded P1 ISPs after the last wash, 1 μl of each of thepre-seeded P1 ISPs was diluted in 99 μl Annealing Buffer and analyzedusing a Guava easyCyte Flow Cytometer. The NTC P1 ISPs were alsoanalyzed for relative size in the absence of bound template.

Determining the Numbers of Copies on the Pre-Seeded P1 ISPs

Based on the counts from the Guava easyCyte Flow Cytometer of thesamples after washing, dilutions were made of the pre-seeded P1 ISPs togive 50,000, 5,000, 500, or 50 ISPs/μl. These dilutions were used in aqPCR reaction as follows: 10 μl Fast SYBR (Thermo Fisher Scientific,Waltham, Mass.), 0.2 μl 10 μM Truncated PCR A Primer (5′ CCA TCT CAT CCCTGC GTG TC 3′; SEQ ID NO: 2), 0.2 μl 10 μM Truncated PCR B Primer(5′-CCT ATC CCC TGT GTG CCT TG-3′; SEQ ID NO: 3), 2 μl pre-seeded P1ISPs, and 7.6 μl NF H₂O. The reaction mixes were placed in a real-timePCR instrument for a denaturation step of 95° C. for 20 sec followed by40 cycles of 95° C. for 3 sec and 60° C. for 30 sec. The C_(t) of eachqPCR reaction was compared to C_(t) values of qPCR reactions with knownnumbers of molecules to calculate the number of copies of monoclonaltemplate on each ISP. Samples with the same amount of templatepre-seeded were combined to obtain an average number of copies per ISPfor each group.

Templating Reaction

The pre-seeded P1 ISPs with the same number of copies of each monoclonaltemplate were pooled, i.e. the P1 ISPs that had been pre-seeded with 70copies of OFA 1 AB, OFA 12 AB, or OFA 13 AB were all pooled andsimilarly the P1 ISPs pre-seeded with 665 copies of the templates werepooled and the P1 ISPs pre-seeded with 4,170 copies of the templateswere pooled. The pools of pre-seeded P1 ISPs (100 μl containing˜375,000,000 pre-seeded P1 ISPs) were combined with 4 μl Primer Mix S(Ion PGM™ Template IA 500 Kit, ThermoFisher Scientific) in solution notattached to any substrate, wherein the template nucleic acid moleculesinclude a primer binding site for the second primer at or near theterminus opposite the proximal segment) and 146 ISP Dilution Buffer,both from the Ion PGM™ Template IA 500. The NTC P1 ISPs (100 μlcontaining 375,000,000 ISPs) were combined with 4 μl Primer Mix S, 131μl ISP Dilution Buffer, and three 5 μl aliquots of the library. Two IonPGM™ Template IA (isothermal amplification) pellets were rehydrated,each with 720 μl Rehydration Buffer, vortexed, and briefly spun.Dehydrated Template IA pellets contained T7 polymerase, uvsXrecombinase, uvsY recombinase loading protein, gp32 protein, Bsu DNApolymerase, dNTPs, ATP, thioredoxin, phosphocreatine, and creatinekinase. Four pellets from a TwistAmp™ Basic kit were rehydrated in 120μL of Rehydration Buffer (25 mM Tris, pH 8.3; 5 mM DTT; 3 mM dNTP;3.5625% Trehalose; 0.1 mg/ml Creatine Kinase; 1.1375 mg/ml Twist gp32;0.4 mg/ml UvsX; 0.1 mg/ml UvsY; 0.25 mU/ul PPiase; 0.02 mg/ml Sau Pol;0.03063 mg/ml T7 Dbl Exo-Pol; 0.0225 mg/ml Thioredoxin (5×); 1.425% PEG35) supplied from the kit (tube 2). The recombinase solution wasvortexed and spun, then iced. To each pool of pre-seeded P1 ISPs and theNTC ISPs, 360 μl of the rehydrated pellet solution were add. Thesolutions were thoroughly vortexed and briefly spun. The templatingreaction was performed at 40° C. for 30 minutes. To stop the templatingreaction, 650 μl of 100 mM EDTA was added, the solution was vortexed,and the tubes were briefly spin.

Enriching the Templated ISPs

The templated ISPs were enriched using MyOne beads (ThermoFisherScientific, Waltham, Mass.). Briefly, each stopped templating reactionwas split into 2 tubes and 100 μl of MyOne beads were added to eachtube. The tube was rotated for 15 minutes at room temperature, spun, andplaced on a magnetic tube rack. After the beads were fully pelleted, thesupernatant was removed and discarded. Then, like samples were pooledusing 500 μl 3 mM SDS solution per tube such that each like-sample sethad a total of 1 ml 3 mM SDS. The tubes were vortexed thoroughly,briefly spun, and placed on a magnetic tube rack for 2 minutes. Thesupernatant was removed and the tubes were removed from the magnetictube rack. The beads were resuspended with 200 μl of melt-off solution(125 mM NaOH and 0.1% Tween 20), vortexed thoroughly, and briefly spun.After a 2 minute room temperature incubation, the bead solution wasplaced on a magnetic tube rack for 2 minutes and the supernatant wastransferred to a new 0.2 ml tube. The tube was spun for 8 minutes atmaximum speed and the supernatant was removed to leave ˜10-15 μlsolution. 90 μl water was added to the templated ISPs and 2 μl wasremoved for analysis on a Guava easyCyte Flow Cytometer. The tube wasspun for 5 minutes at 15,000 rcf and the supernatant was removed toleave 10 μl of solution containing the templated ISPs. 20 μl of 100%PBST and 20 μl of sequencing primer were added to the ISPs and the tubeswere vortexed and spun briefly. The sequencing primer was annealedaccording to the manufacturer's instructions using the Ion Torrent PI™Sequencing HiQ 200 Kit (Life Technologies, Carlsbad, Calif.).

Sequencing

A standard sequencing reaction was conducted on an Ion Torrent PGMaccording to the manufacturer's instructions using an Ion Torrent PI™Sequencing HiQ 200 Kit (Life Technologies, Carlsbad, Calif.). Thesequencing signals were analyzed by Torrent Suite Software to determinethe sequence present within the amplicon of these ISPs.

Results

Pre-seeding the P1 ISPs with monoclonal templates before Bulk IAgenerated better sequencing metrics than similar reactions withoutpre-seeding (i.e. NTC P1 ISPs). Furthermore, the P1 ISPs pre-seeded withmore copies of the monoclonal templates up to 4170, the largest numbertested in this example, had better sequencing metrics than ISPspre-seeded with fewer copies. For example, read length histogramsillustrated that the pre-seeded samples had fewer small length reads(FIGS. 1A-1D). Furthermore, the percentages of ISPs loaded and theusable reads from these loaded ISPs both increased with higher numbersof copies pre-seeded onto ISPs (FIGS. 2A-2B). The total reads and meanand median of the read lengths also increased with number of copiespre-seeded (FIGS. 2C-2D). Negative metrics of sequencing were reducedwith pre-seeding in a copy number dependent manner. For example, thepercentages of empty wells and low quality wells were reduced withpre-seeded P1 ISPs (FIGS. 2E-2F). Overall, this experiment demonstratesP1 ISPs can be pre-seeded a monoclonal template before Bulk IA toprovide significant improvements in next-generation sequencing.

Example 2 Pre-Seeding ISPs with a Single-Cycle PCR Followed byIsothermal Amplification

As in the previous example, this example provides a method to pre-seedISPs with monoclonal templates before an isothermal amplification(templating reaction) for downstream next-generation sequencing.However, in the method disclosed in this example, the templatingreaction was performed after pre-seeded ISPs were distributed into wellsof an Ion Torrent chip. The pre-seeded ISPs were compared to no templatecontrol (“NTC”) ISPs, which had the same template polynucleotides insolution during the templating reaction instead of before the templatingreaction and during the pre-seeding reaction. Sequencing results fromthe different amplification reactions were compared using variousmetrics.

Pre-Seeding Reaction

Accordingly, ISPs with P1 adapters were pre-seeded with OFA 1 AB, OFA 12AB, or OFA 13 AB template nucleic acid molecules as provided inExample 1. After pre-seeding, the pre-seeded ISPs were washed andcounted as provided in Example 1. The pre-seeded ISPs with the samenumber of copies of each monoclonal template were pooled, i.e. the ISPsthat had been pre-seeded with 82 copies of OFA 1 AB, OFA 12 AB, or OFA13 AB were all pooled and similarly the ISPs pre-seeded with 775 copiesof the templates were pooled and the ISPs pre-seeded with 5,400 copiesof the templates were pooled.

Cassette Loading

Ion Torrent 541 chips were washed with 100 μl of 100 mM NaOH for 60seconds, rinsed with 200 μl nuclease-free water, rinsed with 200 μlisopropyl alcohol, and aspirated dry. To load the chip, ISPs(500,000,000 NTC ISPs or pooled, pre-seeded ISPs) were vortexed, broughtto 45 μl with Annealing Buffer (Ion PI™ Hi-Q™ Sequencing 200 Kit, IonTorrent), and injected into the treated chip through the loading port.The chip was centrifuged for 2 minutes at 1424 rcf. 1 ml of foam (980 μl50% Annealing Buffer with 20 μl 10% Triton X-100 were combined, 1 ml ofair was pipetted in, and foam was further mixed by pipette for 5seconds) was injected into the chip and the excess was aspirated. 200 μlof a 60% Annealing Buffer/40% isopropyl alcohol flush solution wasinjected into the chip and the chip was aspirated to dryness. The chipwas rinsed with 200 μl Annealing Buffer and the chip was vacuumed dry.For the chips with pre-seeded ISPs, 40 μl of PBST was added to the chipand each port was filled with 35 μl PBST. For the chip with NTC ISPs, 5μl of 100 μM library (equal parts OFA 1 AB, OFA 12 AB, and OFA 13 AB)was added to 110 μl Annealing Buffer to make a library mix. 40 μl of thelibrary mix was added to the chip and each port was filled with 35 μllibrary mix. The chips were placed on a thermocycler and cycled one timeat 95° C. for 1 minute, then 37° C. for 2 minutes, then 4° C. The chipswere rinsed once with 200 μl Annealing Buffer and left wet.

Templating Reaction

An Ion PGM™ Template IA Pellet was rehydrated with 871 μl of Ion PGM™Rehydration Buffer, vortexed thoroughly, and spun briefly. Each chip wasinjected with 40 μl pellet IA solution and the displaced AnnealingBuffer was aspirated from the exit port. 20 μl pellet IA solution wasadded to the loading port and the chips were spun for 2 minutes at 1424rcf. To activate the pellet IA solution, 218.2 μl Ion PGM™ StartSolution was combined with 8 μl Primer A and added to the pellet IAsolution. The activated pellet IA solution was vortexed and spunbriefly. To each chip, 60 μl of activated pellet IA solution wasinjected and the displaced fluid was aspirated. Each chip had anadditional 35 μl of pellet IA solution added to each port. The chipswere placed on a thermocycler set to 40° C., covered, and incubated for15 minutes. The chips were rinsed with 200 μl 0.5 M EDTA under vacuumand aspirated dry. The chips were rinsed with 200 μl Annealing Bufferunder vacuum and aspirated dry. The chips were rinsed twice with 200 μl1% SDS under vacuum and aspirated dry. The chips were rinsed with 200 μlFlush solution (50% isopropyl alcohol/50% Annealing Buffer) under vacuumand aspirated dry. Then the chips were rinsed with 200 μl AnnealingBuffer and aspirated dry. To each chip 40 μl primer mix (250 μlSequencing Primer and 250 μl Annealing Buffer) was injected into theflow cell and 35 μl primer mix were added to each port. The chips wereplaced on a thermocycler and cycled one time at 95° C. for 2 minutes,then 37° C. for 2 minutes, then 4° C. The chips were rinsed once with200 μl Annealing Buffer and aspirated. To each chip, 60 μl Enzyme Mixwas added (60 μl Annealing Buffer and 6 μl PSP4 enzyme) and incubatedfor 5 minutes. The chips were vacuum dried and 100 μl Annealing Bufferwas added immediately. The chips were loaded onto the Ion Proton Systemfor sequencing.

Sequencing

The Ion Proton System was initialized with Hi-Q 200 materials andsequencing was performed using 400 flows.

Results

Pre-seeding the ISPs with monoclonal templates and then loading thepre-seeded onto an Ion Torrent sequencing chip generated bettersequencing metrics than the NTC ISPs, which had monoclonal templatesattached and amplified in one reaction. The percentage of ISP loadedincreased with 82 copies of template pre-seeded onto the ISPs althoughhigher levels of pre-seeding decreased the percentage of ISP loadedbelow the NTC ISPs (FIG. 3A). The percentage of usable reads increasedwith the pre-seeded ISPs (FIG. 3B). The pre-seeded ISPs also had up to a9-fold increase in total reads relative to the NTC ISPs and greater than2-fold increases in the mean, median, and mode of the read lengths(FIGS. 3C-3D). All the pre-seeded ISPs showed lower percentages of lowquality wells and wells with no template (FIGS. 3E-3F). This exampledemonstrates that ISPs can be pre-seeded with monoclonal templatenucleic acid molecules in a method where templating is performed on ISPsdistributed within the wells of an Ion Torrent chip, to achieve improvedsequencing data.

Example 3 Pre-Seeding ISPs with a Complex Library in an RPA Reaction

As in the previous example, this example provides a method to pre-seedISPs. However, in the method disclosed in this example the templatingreaction was done using a short RPA reaction instead of a single PCRcycle. Furthermore, the reaction was performed on ISPs in the wells ofan Ion Torrent 541 Chip. After the RPA pre-seeding reaction, thepre-seeded ISPs were washed before a second isothermal RPA reaction (atemplating reaction) was performed. The ISPs generated with a two-stepprocess of pre-seeding ISPs followed by a templating reaction wheretemplate nucleic acid molecules were washed away before the templatingreaction, were compared to ISPs that underwent a single RPAamplification without washing, which had the same templatepolynucleotides in the reaction mixture for the entire incubation. Fourreplicates of each of the one-step and two-step reactions were performedand compared.

Pre-Seeding Reaction

Accordingly, 500,000,000 ISPs were added to the wells of an Ion TorrentChip and were pre-seeded with 160,000,000 copies of 130 base pairfragments of the E. coli genome with adapters on the ends of thefragments (A-B, A-AV5, or A-AV6) and the other components in thepre-seeding reaction mixture (same as in Example 2). The adaptersincluded primer binding sites that facilitated binding to an immobilizeduniversal primer (primer B, AV5, or AV6) attached to the ISPs, auniversal primer in solution (primer A) during the pre-seeding andtemplating reaction, and sequencing primers. Briefly, 40 μl of thepre-seeding reaction mixture was added to the chip and each port wasfilled with 35 μl of pre-seeding reaction mixture. The pre-seedingreaction was incubated for 2.5 minutes at 40° C. and then stopped byrinsing with 200 μl 0.5 M EDTA under vacuum and then aspirated dry. Thepre-seeded ISPs were washed with Annealing Buffer.

The templating reactions for the NTC ISPs were assembled as above andincubated at 40° C. incubation for 30 minutes. The NTC ISPs wereprocessed in the same fashion as the pre-seeded ISPs after thetemplating reaction.

Templating Reaction

The templating reaction was performed as in Example 2. The templatingreaction for the pre-seeded ISPs incubated for 30 minutes at 40° C.

Sequencing

Sequencing was performed using an Ion Torrent PI™ Sequencing HiQ 200 Kitas in Example 1.

Results

Pre-seeding the ISPs with template nucleic acid molecules and thentemplating the attached template nucleic acid molecules in a secondreaction (i.e. a templating reaction) generated an average of 7,750,000more reads than the templated ISPs generated without a separatepre-seeding reaction. Furthermore, other sequencing metrics were betterwith a pre-seeding reaction. For the one-step and two-step (separatepre-seeding followed by templating) reactions, the mean AQ20 scores were109 and 110.75 and the mean AQ20GBases scores were 2.375 and 3.075,respectively. This example demonstrates that ISPs pre-seeded with acomplex population of template nucleic acid molecules improvessequencing data generated on the template.

Example 4 Pre-Seeding ISPs with a Complex Library in a Bulk IsothermalAmplification

As in the previous example, this example provides a method to pre-seedISPs with a complex population of template nucleic acid molecules.However, this example provides a pre-seeding RPA reaction with a longerincubation. After the pre-seeding reaction, the pre-seeded ISPs wereenriched using MyOne magnetic beads before a second isothermal RPAreaction (i.e. templating reaction) was performed. After the templatingreaction, the templated ISPs were further enriched using MyOne magneticbeads before being processed for downstream sequencing. The ISPsgenerated with a two-step process of pre-seeding ISPs followed by atemplating reaction where template nucleic acid molecules were washedaway before the templating reaction, were compared to ISPs thatunderwent a single RPA amplification without washing, which had the sametemplate polynucleotides in the reaction mixture for the entireincubation.

Pre-Seeding Reaction

A pre-seeding reaction with a mix of A′ primers and a blocked P1 primerwas performed (see FIG. 4). A tube comprising 50 μl of an AnnealingBuffer (Ion PGM™ Hi-Q™ View Sequencing Solutions, (Part No. A30275)),Primer Mix S (a mix of 3 A′ primers: 5′-ACG ATC CAT CTC ATC CCT GCG TGTC-3′ (SEQ ID NO: 4); 5′-TCC ATA AGG TCA GTA ACG ATC CAT CTC ATC CCT GCGTGT-3′ (SEQ ID NO: 5); and 5′-/5-Bio/TCC ATA AGG TCA GTA ACG ATC CAT CTCATC CCT GCG TGT-3′ (SEQ ID NO: 5)), a blocked fusion primer (5′-CCT ATCCCC TGT GTG CCT TGG CAG TCT CAG CCA CTA CGC CTC CGC TTT CCT CTC TAT GGAA/3-Phos/-3′; SEQ ID NO: 6), 7.5×10⁶ ISPs, and 35 pM 130 bp templatenucleic acid molecules from a human genome library was vortexed, brieflyspun and incubated at 98° C. for 2 minutes, and then incubated at 37° C.for 2 minutes. Another aliquot of Annealing Buffer was added (75 μl) andthe solution was transferred to a new tube and vortexed. An Ion PGM™Template IA Pellet (ThermoFisher Scientific, Waltham, Mass.) wasrehydrated with 720 μl Ion PGM™ Template IA Rehydration Buffer,thoroughly mixed, and stored on ice. 375 μl of the rehydrated Ion PGM™Template IA Pellet were combined with the previously prepared 125 μlsolution containing the library, vortexed, then placed back on ice. Themixture was combined with 150 μl of pre-mixed Ion PGM™ Template IA StartSolution, thoroughly mixed to form the pre-seeding reaction mixture,briefly centrifuged, and placed on ice. To initiate the pre-seedingreaction, the tube was placed in a 40° C. heat block and then incubatedfor 4 minutes at 40° C. to generate pre-seeded ISPs. The pre-seedingreaction was stopped by the addition of 650 μl of 100 mM EDTA followedby vortexing.

A control reaction was assembled as above, with the followingdifferences: 5.625×10⁶ ISPs and 11 pM template were used and the 40° C.incubation was maintained for 30 minutes. These control reactions wereprocessed in the same fashion as the pre-seeded samples subsequent toenriching the templated ISPs.

Enriching the Pre-Seeded ISPs

The pre-seeded ISPs were enriched using MyOne beads (ThermoFisherScientific, Waltham, Mass.). Briefly, 100 μl of MyOne beads were addedto the tube with the pre-seeded ISPs and vortexed. The tube was rotatedfor 10 minutes, spun, and placed on a magnetic tube rack. After thebeads were fully pelleted, the supernatant was removed and discarded.The beads were washed once with 1 ml of Ion PGM™ Wash Solution using themagnetic tube rack. Then the beads were washed once with 1 ml 3 mM SDSsolution using the magnetic tube rack and the supernatant wastransferred to a new tube for further processing. The supernatant fromthe SDS wash was spun for 8 minutes at 21,000 rcf and the supernatantwas removed to leave approximately 50 μl. The pre-seeded ISPs wereresuspended with 150 μl of melt-off solution (125 mM NaOH and 0.1% Tween20) and transferred to a new tube. The tube was spun for 5 minutes at15,000 rcf and the supernatant was removed to leave 10 μl solution. 190μl water was added to the pre-seeded ISPs and 2 μl was removed foranalysis on a Guava easyCyte Flow Cytometer. The tube was spun for 5minutes at 15,000 rcf and the supernatant was removed to leave 10 μl ofsolution containing the remaining pre-seeded ISPs.

Templating Reaction

111 μl of Annealing Buffer and 4 μl of Primer Mix S were added to thetube containing the pre-seeded ISPs. The pre-seeded ISPs were pipettedup and down to combine and the entire solution was transferred to a newtube. 375 μl of a rehydrated Ion PGM™ Template IA Pellet was added tothe tube and the tube was vortexed, briefly spun, and placed on ice. Thereaction mixture was combined with 150 μl of pre-mixed Ion PGM™ TemplateIA Start Solution, thoroughly mixed, briefly centrifuged, and placed onice. To initiate the templating reaction, the tube was placed in a 40°C. heat block and then incubated for 30 minutes at 40° C. The templatingreaction was stopped by the addition of 650 μl of 100 mM EDTA followedby vortexing.

Enriching the Templated ISPs

The templated ISPs were enriched using MyOne beads (ThermoFisherScientific, Waltham, Mass.). Briefly, 100 μl of MyOne beads were addedto the tube with the templated ISPs and vortexed. The tube was rotatedfor 10 minutes, spun, and placed on a magnetic tube rack. After thebeads were fully pelleted, the supernatant was removed and discarded.The beads were washed once with 1 ml of Ion PGM™ Wash Solution using themagnetic tube rack. 200 μl of melt-off solution (125 mM NaOH and 0.1%Tween 20) was added to the tube, mixed, and the solution was transferredto a new tube. The tube was spun for 5 minutes at 15,000 rcf and thesupernatant was removed. The ISPs were washed with 200 μl water and 2 μlwas removed for analysis on a Guava easyCyte Flow Cytometer. The ISPswere then processed for sequencing according to the Proton Hi-Q userguide.

Results

Pre-seeding the ISPs with template nucleic acid molecules and thentemplating the attached template nucleic acid molecules in a secondreaction (i.e. a templating reaction) generated an average of 9,167,000more reads than the templated ISPs generated without a separatepre-seeding reaction. Furthermore, other sequencing metrics were betterwith a pre-seeding reaction. For the one-step and two-step (separatepre-seeding followed by templating) reactions, the mean AQ20 scores were105 and 110.25 and the mean AQ20GBases scores were 2.889 and 4.267,respectively. This example demonstrates that ISPs pre-seeded with acomplex population of template nucleic acid molecules yield improvedsequencing data for the template.

Example 5

Seeding

A library (2.4B copies) was mixed with biotin TPCRA (1 uL at 100 uM) ina PCR tube. The tube is filled to 20 uL with 1× Platinum HiFi mix. Thetube was thermo cycled on a thermocycler one time (2 min at 98 C, 5 minat 37 C, 5 min at 54 C). 6 billion beads were added to the tube from. 1×HiFi was added to increase volume by 50% (i.e. 20 uL of beads+10 uL ofPlatinum Hifi mix). The solution was thermo cycled on a thermocycleronce (2 min at 98 C, 5 min at 37 C, 5 min at 54 C).

1 mL myOne beads are pipetted into a 1.5 mL tube (1 mL myOne beads usedfor 2 samples) and the tube was put on a magnet and the supernatantdiscarded. 1 mL 3% BSA in 1×PBS is added to the MyOne mixture, vortexed,pulse spun. The mixture was put on a magnet and the supernatantdiscarded. 1 mL AB is added to MyOne mixture, vortexed, pulse spun. Themixture was put on a magnet and the supernatant discarded. 250 uL AB isadded to the MyOne mixture (one sample uses 125 uL 4× concentratedMyOnes). The purified MyOne mixture was transferred to new 1.5 mL tube.

Samples from the PCR tube were transfer to new 1.5 mL tube. 125 uL 4×concentrated MyOnes were added to the ISP mix. The mixture was pipettedup and down 3 times (200 uL/s) and let sit for 10 min. The mixture wasput on a magnet, MyOne captured ISP were pulled out (chef speed 80 uL/s)and the supernatant was discarded. 20 uL NF water was added, pulsevortexed, pulse spun, and put on magnet to pellet MyOne.

Chip Preparation

A chip was rinsed 2× with 200 μL NF water.

Magnetic ISP Loading

20 ul of ISP mixture was mixed with 4.5 uL 10× annealing buffer and 20.5uL water (total 45 ul). ISPs were vortexed and combined with 10×annealing buffer and water. The ISP solution was vortexes and quickspun. The ISP solution was slowly injected into the chip through theloading port. Magnetic loading was performed for 40 minutes at 30sec/sweep. 200 μL of foam (0.2% Triton in 1× AB) was injected throughthe chip, and the excess is extracted. While vacuuming exit port, 200 μL1× AB was added and then aspirated to dry chip. While vacuuming exitport, 200 μL Flush (60% AB/40% IPA) was aspirated and then aspirated todry chip. While vacuuming exit port, 200 μL 1× AB was added. The chip iskept in 1× AB until ready to amplify ISPs on chip.

Amplification—Keep all Reagents on Ice

1st Step Amplification

A tube with biotinylated primer A and blocking molecule (Neutravidin)was prepared and incubated on ice for >15 minutes. Solutions include 1.1uL 100 uM primer per chip and 1 uL 10 mg/mL NAv (rehydrated in 0-PEGbuffer) per chip. 871 μL of Rehydration buffer was added to 1× IA pellet(lot LTBP0047, PN 100032944). The solution was pulse vortexed 10×, quickspun to collect tube contents. The contents were split into two tubes ofequal volume (Put 900 uL in separate tube). One tube of 900 μL was usedfor 1st step amplification, save other tube of 900 μL for 2nd stepamplification.

For each chip to be run, 60 μL pellet solution was slowly injected intothe chip. The displaced annealing buffer was aspirated from exit port.The chip was incubated with pellet solution at RT for 4 minutes. 177.4μL start solution was added to tube of pellet solution, pulse vortexed10× and quick spun. 110 uL/chip of starter solution was transferred totube of primer and blocker, pulse vortexed 10× and quick spun. For eachchip, ˜60 μL activated pellet solution was slowly injected into thechip. All displaced fluid was aspirated from both ports. 25 μL pelletsolution was added to each port. Chips were placed onto hot plate(thermocycler) set to 40° C. The chips were covered with pipette tip boxlid or similar (not the heated thermocycler cover) and let incubate for2.5 minutes.

Short Reaction Stop and Clean Between Amplification Steps

Amplified chips were placed near hood equipped with vacuum. Whilevacuuming exit port, 200 μL 0.5 M EDTA pH 8 (VWR E522-100ML) was addedthen aspirated to dry the chip. While vacuuming exit port, 200 μL 1× ABwas aspirated and then aspirate to dry the chip. The addition of AB wasrepeated and the chip is left wet for 2nd step amplification. (Vac outthe AB twice and leave the 3rd AB in chip)

2nd Step Amplification (No Blocker)

A tube with biotinylated primer A was prepared and incubated on icefor >15 minutes. Solutions include 1.1 uL 100 uM primer per chip. 871 μLof Rehydration buffer was added to 1× IA pellet (lot LTBP0047, PN100032944). The solution was pulse vortexed 10×, quick spun to collecttube contents. After discarding appropriate volume of pellet solution,6.6 μL 100 uM biotinylated primer was added to pellet mix and it waspulse vortexed 10×.

177.4 μL start solution was added to tube of pellet solution, pulsevortex 10× and quick spin. For each chip, ˜60 μL activated pelletsolution was injected into the pre-spun chip. Displaced fluid wasaspirated from both ports. An additional 25 μL pellet solution was addedto each port. Chips were placed onto hot plate (thermocycler) set to 40°C. The chips were covered with pipette tip box lid or similar (not theheated thermocycler cover) and let incubate for 20 minutes.

Reaction Stop and Clean Up

Amplified chips were placed near hood equipped with vacuum. Whilevacuuming exit port, 200 μL 0.5 M EDTA pH 8 was added and the chips areaspirated to dry chip. While vacuuming exit port, 200 μL 1× AB) wasadded and then aspirated to dry chip. While vacuuming exit port, 200 μL1% SDS solution in water (Ambion PN AM9822) was added and then aspiratedto dry chip. The SDS wash is repeated. While vacuuming exit port, 200 μLformamide was added. The chip was incubated 3 minutes at 50 C, thenaspirated to dry the chip. While vacuuming exit port, 200 μL Flush (50%IPA/50% AB) solution was added. The chip was aspirated to dry. Whilevacuuming exit port, 200 μL annealing buffer was added. The chip wasleft in 1× AB until ready for priming.

On Chip Sequencing Primer Hybridization and Enzyme

Sequencing primer tube was thawed. Primer mix of final 50%/50% AB/primermixture was prepared and vortexed well. If tube of sequencing primer hasa volume of 250 μL, 250 μL 1×AB was added. The chip was aspirated to drythen 80 μL primer mix was added to the chip (50 μL in flow cell, 30 μLin ports). The chip was placed on thermocycler & incubated at 50° C. for2 min, 20° C. for 5 min. 200 μL 1× AB was injected while vacuuming exitport. The enzyme mix was prepared with 60 μL annealing buffer & 6 μLPSP4 enzyme. The ports were cleaned and vacuumed to dry chip from theinlet port. 60 μL enzyme mix was added to the chip and incubated at RTfor 5 minutes. The chip was aspirated to dry the chip from the inletport. 100 μL of 1× AB was added to the chip immediately. The ports werecleaned, the back of the chip was dried, and the chip was loaded on theProton for sequencing.

Example 6

Seeding

An Ampliseq Exome library (2.4B copies) with A and B adapters was mixedwith a 5′-biotinylated primer complimentary to the A adapter, TPCRA, (1uL at 100 uM) in a PCR tube. The tube was filled to 20 uL with 1×Platinum HiFi mix containing Taq DNA polymerase high fidelity, salts,magnesium and dNTPs. The tube was thermo cycled on a thermocycler onetime (2 min at 98° C., 5 min at 37° C., 5 min at 54° C.). Ion SphereParticle (ISP) beads (6 billion), each having thousands of B primerimmobilized thereto, were added to the tube. 1× HiFi was added toincrease volume by 50% (i.e. 20 uL of beads+10 uL of Platinum Hifi mix).The solution was thermo cycled on a thermocycler once (2 min at 98° C.,5 min at 37° C., 5 min at 54° C.).

In an alternative method, in a PCR tube, 1.2 billion copies of IonAmpliseq Exome library (20 μL 100 pM, with standard Ion Torrent A and P1library adapters) was mixed with 3 μL 3 μM biotin-TPCRA (sequence5′biotin-CCA TCT CAT CCC TGC GTG TC-3′; SEQ ID NO: 2) and 3 μL 1.5 μMB-trP1 (trP1 is a 23mer segment of the Ion P1 adapter with sequence CCTCTC TAT GGG CAG TCG GTG AT (SEQ ID NO: 1); B is the ISP primer sequence)primers, and 9 μL Ion Ampliseq HiFi Master Mix 5×. The volume was filledup to 45 μL with 10 μL nuclease-free water. The tube was thermocycled ona thermocycler with the following temperature profile: 2 min at 98° C.,2 cycles of [15 sec at 98° C.-2 min at 58° C.], final hold at 10° C.After the thermocyling, 6 billion ISPs (75 μL 80 million/μL), and 6 IonAmpliseq HiFi Master Mix 5× were added to the tube. 5 μL nuclease-freewater was also added to bring up total volume to 131 μL. The solutionwas mixed well and the tube was returned to the thermocycler. A thirdcycle of amplification was performed with the following temperatureprofile: 2 min at 98° C., 5 min at 56° C., final hold at 10° C. Afterthermocycling, add 5 μL EDTA 0.5M and mix to stop the reaction

Enriching of the ISPs

MyOne superparamagnetic beads (1 mL) with streptavidin covalentlycoupled to the bead surface were pipetted into a 1.5 mL tube (1 mL MyOnebeads used for 2 samples) and the tube was put on a magnet and thesupernatant discarded. 1 mL 3% BSA in 1×PBS was added to the MyOnemixture which was then vortexed and pulse spun. The mixture was put on amagnet and the supernatant discarded. Annealing buffer (AB; 1 mL) wasadded to the MyOne mixture, vortexed and pulse spun. The mixture was puton a magnet and the supernatant discarded. AB (250 uL) was added to theMyOne mixture (one sample uses 125 uL 4× concentrated MyOnes). Thepurified MyOne mixture was transferred to a new 1.5 mL tube.

Samples from the PCR tube containing the ISP mix were transferred to anew 1.5 mL tube. Concentrated (4×) MyOne beads (125 uL) were added tothe ISP mix. The mixture was pipetted up and down 3 times (200 uL/s) andthen allowed to sit for 10 min. The mixture was put on a magnet,MyOne-captured ISPs were pulled out (chef speed 80 uL/s) and thesupernatant was discarded. Nuclease-free (NF) water (20 uL) was added tothe tube which was then pulse vortexed, pulse spun, and put on magnet topellet the MyOne beads.

In an alternative method of enriching the ISPs, 120 μL of MyOneStreptavidin C1 beads were transferred into a separate tube and the tubewas placed on a magnet to pellet the magnetic beads. The supernatant wasdiscarded and the tube was removed from the magnet. The beads werewashed by resuspending in 150 μL Ion Torrent Annealing Buffer, thenpelleting on a magnet. The supernatant was discarded, and the wash wasrepeated one more time with 150 μL Annealing Buffer. After discardingsupernatant from the second wash, the washed MyOne C1 beads wereresuspended with 50 μL Annealing Buffer. The whole content of the washedMyOne C1 in Annealing Buffer was transferred to the thermocycled PCRtube containing library and ISPs. The pipette volume was set to 160 μL,and the contents were mixed slowly by pipetting up and down three timesat 1 sec per aspiration or dispensing motion. The mixture was allowed tosit at room temperature for 30 min without agitation to allow magneticbeads to capture library seeded ISPs. The tube was then put on a magnetto pellet magnetic beads and the supernatant was discarded. Tween-20 (25μL 0.1%) in water was added to the pellet. The mixture was vortexedvigorously to elute seeded ISPs from MyOne C1 beads. The tube was pulsespun then returned to magnet. The supernatant (eluent) containing seededISPs was collected in a fresh tube for downstream chip loading andamplification steps.

Chip Preparation

A chip was rinsed 2× with 200 μL NF water.

Magnetic Loading of ISPs onto Chips

Several methods of preparing the ISP/library mixture and loading it ontoan Ion Torrent semiconductor chip containing reaction chamber microwellswere used. In one method, the ISP/Library mixture (20 ul) was mixed with4.5 μl 10× annealing buffer and 20.5 μl water (total 45 μl). The mixturewas vortexed and spun. The ISP solution was slowly injected into thechip through the loading port. Magnetic loading was performed for 40minutes at 30 sec/sweep. A foam (200 μL) containing 0.2% Triton in 1× ABwas injected through the chip, and the excess was extracted. Whilevacuuming the exit port of the chip, 200 μL 1× AB was injected into thechip and then aspirated to dry the chip. While vacuuming exit port, 200μL Flush (60% AB/40% IPA) was then injected into the chip and thenaspirated to dry chip. While vacuuming exit port, 200 μL 1× AB was thenadded by injection into the chip. The chip was kept in 1× AB until readyto amplify the nucleic acids on the ISPs on the chip.

In another method, 150 μL Dynabeads M-270 streptavidin (Thermo FisherScientific), which are magnetic beads with streptavidin bound to thesurface thereof, were transferred to a tube which was then placed in amagnet to pellet magnetic beads. The supernatant was discarded and thetube was removed from the magnet. The following was then added to thetube containing the M-270 pelleted beads: 20 μL ISP mixture from theseeding process, 9 μL 5× Annealing Buffer, and 16 μL nuclease-free waterfor a total 45 μL. Alternatively, 20 ul of ISP/Library mixture was mixedwith 3.2 uL 10× annealing buffer 3 uL concentrated M270 magnetic beadsand 5.8 uL water for a total of 32 ul. The mixture was mixed toresuspend the M-270 pellet, and slowly injected into the chip throughthe loading port. A magnet placed beneath the chip was swept across thechip back and forth repeatedly to load ISPs into chip microwells. Themagnetic loading sweeping was performed for 40 minutes at 30 sec/sweep.After loading, a 15 mL falcon tube containing 5 mL 1% SDS was vigorouslyshaken to generate a dense foam, 800 μL of which was then injectedthrough the chip to remove magnetic beads from the chip flow cell. Flowthrough at the chip exit was discarded. Annealing Buffer (200 μL) wasthen injected through the chip, and the flow through was discarded. Thechip was vacuumed dry from the chip exit. Flush (200 μL of 60% AnnealingBuffer, 40% IPA) was injected through the chip which was then vacuumeddry. Annealing Buffer (200 μL) was injected to fill the chip flow cell,and the flow through was discarded at the chip exit. The chip was leftfilled with Annealing Buffer until ready to amplify in downstreamamplification steps.

Amplification

First Step Amplification

For each chip being amplified, 1.1 uL biotinylated primer A (100 uM) and1 uL blocking molecule (10 mg/mL Neutravidin rehydrated in buffer) werecombined in a tube and incubated on ice for >15 minutes.

Rehydration buffer (871 μL) was added to 1× IA pellet (PN 100032944)containing reaction components for conducting recombinase-polymeraseamplification (e.g., recombinase, polymerase, single-stranded bindingprotein, nucleotides, buffers and other ingredients) from the ION PGM™TEMPLATE IA 500 kit. The solution was pulse vortexed 10× and quick spunto collect tube contents. The rehydrated contents (referred to as“pellet solution”, at roughly 900 ul) were kept on ice during theprocess.

For each Ion Torrent chip, 60 μL of rehydrated IA pellet solution wasslowly injected into the chip. The displaced annealing buffer wasaspirated from the exit port. The chip was incubated with rehydrated IApellet solution at RT for 4 minutes.

For each chip being amplified, 90 uL of rehydrated IA pellet solutionwas transferred to a new tube. The previously prepared biotinylatedprimer A and neutravidin blocking molecule (2.1 uL) was added and pulsemixed. A start solution (30 μL), containing an aqueous solution of 28 mMMg(OAc)₂, 10 mM Tris acetate and 3.75% (V/V) methyl cellulose, was addedto the tube of rehydrated IA pellet solution, pulse vortexed 10× andquick spun to form an activated amplification solution in a ˜120 uLtotal volume. For each chip, ˜60 μL activated amplification solution wasslowly injected into the chip. All displaced fluid was aspirated fromboth ports. Next, 25 μL of remaining activated amplification solutionwas added to each chip port. Chips were placed onto a hot plate(thermocycler) set to 40° C. The chips were covered with a pipette tipbox lid or similar cover (not the heated thermocycler cover) and allowedto incubate for 2.5 minutes.

Short Reaction Stop and Clean Between Amplification Steps

Amplified chips were taken off the hot plate or thermocycler. Whilevacuuming the exit port, 200 μL 0.5 M EDTA pH 8 (VWR E522-100ML) wasinjected into the chip and the chip was then aspirated to dry using avacuum. While vacuuming the exit port, 200 μL 1× AB was injected intothe chip which was then aspirated to dry. The addition of AB wasrepeated two more times and the chip was left filled for the 2nd stepamplification. (The AB was vacuumed out twice and the third addition ofAB was left in the chip.)

Second Step Amplification (No Blocker)

For each chip, 60 uL rehydrated pellet solution was slowing injectedinto the chip. The displaced annealing buffer was aspirated from theexit port. The chip was incubated with pellet solution at RT for 4minutes.

For each chip being prepared, 90 uL of rehydrated pellet solution wastransferred to a fresh tube. Biotinylated Primer A (1.1 uL of 100 uM)was added and the tube pulse vortexed and spun.

Start solution (30 μL) was added to the tube containing rehydratedpellet solution and Primer A and was pulse vortexed 10× and quick spunto generate an activated amplification solution. Approximately 60 μLactivated amplification solution was injected into the chip. Displacedfluid was aspirated from both ports. An additional 25 μL of remainingamplification solution was added to each port. Chips were placed onto ahot plate (thermocycler) set to 40° C. The chips were covered with apipette tip box lid or similar cover and allowed to incubate for 20minutes.

Reaction Stop and Clean Up

Chips that had been subjected to amplification reactions were placednear a hood equipped with a vacuum. While vacuuming the exit port, 200μL 0.5 M EDTA pH 8 was added and the chips were aspirated to dry thechips. While vacuuming the exit port, 200 μL 1× AB was added and thenaspirated to dry the chip. While vacuuming the exit port, 200 μL 1% SDSsolution in water (Ambion PN AM9822) was added and then aspirated to drythe chip. The SDS wash was repeated. While vacuuming the exit port, 200μL formamide was added. The chip was incubated 3 minutes at 50° C., thenaspirated to dry the chip. While vacuuming the exit port, 200 μL Flush(50% IPA/50% AB) solution was added. The chip was aspirated to dry.While vacuuming the exit port, 200 μL annealing buffer was added. Thechip was left in 1× AB until ready for priming.

On Chip Sequencing Primer Hybridization and Enzyme Reaction

A tube containing Ion sequencing primer (100 uM) was thawed. For eachchip being sequenced, a primer mixture of 40 uL annealing buffer and 40uL sequencing primer was prepared and vortexed well. The chip wasaspirated to dry then 80 μL of primer mixture was added to the chip (50μL in flow cell, 15 μL in each port). The chip was placed on athermocycler and incubated at 50° C. for 2 min, 20° C. for 5 min. 200 μL1× AB was injected while vacuuming the exit port. An enzyme mixture wasprepared with 60 μL annealing buffer and 6 μL sequencing enzyme (IonPSP4 Sequencing Polymerase). The ports were cleaned and vacuumed to drythe chip from the inlet port. Enzyme mixture (60 μL) was added to thechip and incubated at RT for 5 minutes. The chip was aspirated to dry.AB (100 μL of 1×) was injected to fill the chip immediately. The portswere cleaned, the back of the chip was dried, and the chip was loaded onthe Ion Torrent Proton (Thermo Fisher Scientific) apparatus forsequencing of the library nucleic acids.

Example 7 Comparison of Sequencing Results Using Different Nucleic AcidManipulation Methods

FIG. 10 shows the total usable reads for groups of nucleic acidsequencing runs of nucleic acid templates generated using four differentamplification conditions (A-D in the figure). In method A, non-templatedIon Sphere Particles (ISPs) were loaded into microwells of Ion Torrent541 chips according to methods described in Example 2 herein.Subsequently, library molecules (a 110-bp hg19 fragment library) withadaptors complimentary to the ISP primer were hybridized to thepre-loaded ISPs with a single 95° C. 1 min/37° C. 1 min thermocyclingstep following injection of the library amplicons into the chip.Following library hybridization, amplification was performed using asingle-step RPA templating amplification method essentially as describedin Example 2. The primers were not biotinylated. Method B employed twoimportant changes to amplification protocol A: 1) an additionalamplification step (a “first” amplification step) prior to thetemplating amplification and 2) incorporation of neutravidin andbiotinylated solution primers in the added first amplification step. Inthis example, the first amplification step, which is an isothermal RPAamplification, is 2.5 minutes and contains an equivalent concentrationof biotinylated solution primer and neutravidin. The secondamplification step is 15 minutes and does not contain neutravidin. Inthis method, the first amplification step serves to locally amplifytemplate copies while adding drag (via neutravidin) to limit well-2-welldiffusion of nascent strands. After 2.5 minutes, enough local copies arecreated that the drag component is no longer needed. The secondamplification step was then carried out as described in Example 2. InMethods C and D, a 220-bp hg19 Ampliseq Exome Library was used. Method Cimproves upon method B by replacing the library hybridization method inmethods A and B, with a solution based pre-amplification ISP enrichmentmethod as described in Example 6 herein. Thus, instead of performing allsteps in wells, the first step of hybridizing the library to the ISPswas done in solution, then magnetic beads were added to the tube, thetemplate-containing ISPs were enriched, and then separated from themagnetic beads and loaded into the wells for the 2-step amplification aswas done in Method B. The pre-amplification enrichment method enablesloading of ISPs with single library template copies. Finally,amplification method D, which was carried out according to Method C,employed a modified ISP primer sequence compared with method C. Themodified primer, AV4, has the following sequence:ATTCGAGCTGTTCATCTGTATCTTGCGCTACCAA (SEQ ID NO: 7). As shown in FIG. 10,the combination of improvements made from Methods A-D enable total readsequivalent to sequencing carried out on template nucleic acids amplifiedthrough emulsion PCR

The disclosed embodiments, examples and experiments are not intended tolimit the scope of the disclosure or to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. It should be understood that variations in the methods asdescribed may be made without changing the fundamental aspects that theexperiments are meant to illustrate.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed. Those skilled in the art can devise manymodifications and other embodiments within the scope and spirit of thepresent disclosure. Indeed, variations in the materials, methods,drawings, experiments, examples, and embodiments described may be madeby skilled artisans without changing the fundamental aspects of thepresent disclosure. Any of the disclosed embodiments can be used incombination with any other disclosed embodiment. When multiple low andmultiple high values for ranges are given, a skilled artisan willrecognize that a selected range will include a low value that is lessthan the high value. All headings in this specification are for theconvenience of the reader and are not limiting. In the foregoingspecification, the concepts have been described with reference tospecific embodiments. However, one of ordinary skill in the artappreciates that various modifications and changes can be made withoutdeparting from the scope of the invention as set forth in the claimsbelow. Accordingly, the specification and figures are to be regarded inan illustrative rather than a restrictive sense, and all suchmodifications are intended to be included within the scope of invention.Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims. After reading the specification,skilled artisans will appreciate that certain features are, for clarity,described herein in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures that are, for brevity, described in the context of a singleembodiment, may also be provided separately or in any subcombination.Further, references to values stated in ranges include each and everyvalue within that range.

What is claimed is:
 1. A method for generating nucleic acid moleculescomprising a specific nucleotide sequence, comprising: (a) obtaining apopulation of nucleic acid molecules in which each molecule comprises afirst sequence of contiguous nucleotides at the 5′ end of the molecule,a second sequence of contiguous nucleotides at the 3′ end of themolecule and a third nucleotide sequence positioned between the firstand second nucleotide sequences, wherein the first nucleotide sequenceand second nucleotide sequence are different from each other, andwherein the first nucleotide sequences of the nucleic acid molecules areidentical and the second nucleotide sequences of the nucleic acidmolecules are identical among the population; and (b) subjecting thepopulation of nucleic acid molecules to two or more cycles of nucleicacid amplification in the presence of one or more forward primerscomprising an oligonucleotide sequence identical to the first nucleotidesequence and a reverse primer that is blocked at the 3′ end andcomprises an oligonucleotide sequence complementary to the secondnucleotide sequence that is linked at the 5′end of the oligonucleotidesequence to a fourth nucleotide sequence that is not complementary tothe second nucleotide sequence to generate nucleic acid products whichcomprise a sequence of nucleotides complementary to the fourthnucleotide sequence.
 2. The method of claim 1, further comprisingexposing single-stranded nucleic acids of the products of (b) to asingle-stranded oligonucleotide that is identical to the fourthnucleotide sequence under annealing conditions thereby hybridizing theproducts of (b) that comprise a sequence of nucleotides complementary tothe fourth nucleotide sequence to the single-stranded oligonucleotidethat is identical to the fourth nucleotide sequence to generate apartially double-stranded oligonucleotide-bound product.
 3. The methodof claim 2, further comprising extending the 3′ end of theoligonucleotide that is hybridized to the product that comprises asequence of nucleotides complementary to the fourth nucleotide sequencethereby generating a double-stranded nucleic acid by synthesizing anucleic acid strand comprising the single-stranded oligonucleotide thatis identical to the fourth nucleotide sequence and having a nucleotidesequence that is complementary to the product to which it is hybridized.4. The method of claim 3, wherein the nucleic acid strand comprising thesingle-stranded oligonucleotide sequence that is identical to the fourthnucleotide sequence is attached to a support at the 5′ end of thestrand, and further comprising isolating the synthesized single-strandednucleic acid strand attached to the support by removing the support fromany other nucleic acids that are not bound to the support.
 5. The methodof claim 4, wherein the support is a solid support bead.
 6. The methodof claim 4, wherein the extending is carried out using arecombinase-polymerase amplification (RPA) reaction.
 7. The method ofclaim 6, wherein the RPA reaction is performed by incubating an RPAreaction mixture for 2 to 5 minutes at a temperature between 35° C. and45° C.
 8. The method of claim 4, wherein the extending is carried outusing a polymerase chain reaction (PCR).
 9. The method of claim 8,wherein the extending includes performing three PCR cycles.
 10. A methodof preparing a device for nucleic acid analysis, the method comprising:generating a template nucleic acid including a capture sequence portion,a template portion, and a primer portion modified with a linker moietyaccording to the method of claim 1, wherein the sequence of nucleotidescomplementary to the fourth nucleotide sequence corresponds to a capturesequence portion, the third nucleotide sequence is a template portion,and the forward primer is modified with a linker moiety and the forwardprimer sequence corresponds to the primer portion; capturing thetemplate nucleic acid on a bead support having a plurality of captureprimers complementary to the capture sequence portion of the templatenucleic acid, the capture primers hybridizing to the capture sequenceportion of the template nucleic acid; linking the captured templatenucleic acid to a magnetic bead having a second linker moiety to form abead assembly, the second linker moiety attaching to the first linkermoiety; and loading the bead assembly into a well of a device fornucleic acid analysis using a magnetic field, wherein the device is asequencing device.
 11. The method of claim 10, further comprisingextending the capture primer complementary to the template nucleic acidto form a sequence target nucleic acid attached to the bead support. 12.The method of claim 11, further comprising denaturing the templatenucleic acid and the sequence target nucleic acid to release themagnetic bead from the bead support.
 13. The method of claim 12, whereindenaturing includes enzymatic denaturing or denaturing in the presenceof an ionic solution.
 14. The method of claim 11, wherein the extendingis carried out using a recombinase-polymerase amplification (RPA)reaction.
 15. The method of claim 14, wherein the RPA reaction isperformed by incubating an RPA reaction mixture for 2 to 5 minutes at atemperature between 35° C. and 45° C.
 16. The method of claim 11,wherein the extending is carried out using a polymerase chain reaction(PCR).
 17. The method of claim 16, wherein the extending includesperforming three PCR cycles.
 18. The method of 12, further comprisingamplifying the sequence target nucleic acid to form a population ofsequence target nucleic acids on the bead support in the well.
 19. Themethod of claim 18, wherein amplifying the sequence target nucleic acidincludes performing recombinase polymerase amplification (RPA), whereperforming RPA includes performing RPA for a first period, washing, andperforming RPA for a second period, the first period shorter than thesecond period.